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The Feulgen reaction: A brief review and new perspectives
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Review
The Feulgen reaction: A brief review and new perspectives Maria Luiza S. Mello
⁎,1
, Benedicto de Campos Vidal1
Department of Structural and Functional Biology, Institute of Biology, University of Campinas (Unicamp), 13083-862 Campinas, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Feulgen reaction DNA Cytochemistry Image analysis Epigenetics
The Feulgen reaction has been proposed by Robert Feulgen and Heinrich Rossenbeck for the identification of DNA nearly a hundred years ago. Since then, many other applications of this cytochemical/topochemical procedure at qualitative and quantitative level have been proposed in relation to DNA and its role in chromatin in human, animal and plant cells. In this article, we briefly review some fundamental aspects of the Feulgen reaction and current applications of such a method in studies of altered chromatin texture, including its association with or preceding changes in transcriptional activities and effect on epigenetic marks. Further perspectives on the use of the Feulgen reaction will depend of the proposal of innovative biological questions in which its reveals appropriate.
1. Basic proposals Feulgen reaction is a classic cytochemical/topochemical method that has allowed investigators to reveal DNA as a component of chromatin and chromosomes. Feulgen-positive images exhibit a magenta (red-purple) color when observed under ordinary light microscopy or a red color under fluorescence microscopy (Fig. 1a–d). Proposed nearly a hundred years ago by Robert Feulgen and Heinrich Rossenbeck (1924), the Feulgen reaction continues to offer an opportunity for many research insights in fields such as cytogenetics, cell biology and analytical cytopathology (Hardie et al., 2002; Biesterfeld et al., 2011; Nielsen et al., 2012), as well as in current chromatin remodeling studies following changes in transcriptional activities (Felisbino et al., 2011, 2014; Natarajan et al., 2012; Poplineau et al., 2013; Vidal et al., 2014; Veronezi et al., 2017). A historical perspective of Robert Feulgen’s life and the development of the cytochemical reaction that was named after him were reported by Kasten (2003). One of the first applications of the Feulgen reaction allowed Alfert, Lison and Pasteels to demonstrate that DNA replication occurs during the S phase of the cell cycle. The constancy of the average DNA content per haploid chromosome set in numerous animal and plant species and the establishment of polyploidy and aneuploidy in particular cell types could also be determined (Swift, 1953; Swift, 1955 − reviews; Chieco and Derenzini, 1999 − review; Hardie et al., 2002). The Feulgen reaction permits the analysis of DNA to occur specifically and stoichiometrically in individual cells using different methods of microspectrophotometric analysis and digital image analysis. It has
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been considered an objective method with high interobserver reproducibility and a useful tool for diagnostic and prognostic purposes (Biesterfeld et al., 2011; Nielsen et al., 2012; Demirel et al., 2013; Poplineau et al., 2013). It even presents some advantages over flow cytometry, such as possible long-term storage of the biological material, visual control for semi-interactive recognition during the analysis, applicability to single cells and feasible determination of very small amounts of DNA (Praça-Fontes et al., 2011). In image analysis studies of chromatin supraorganization, the Feulgen reaction can identify nuclear phenotypes that discriminate regions corresponding to heterochromatin and euchromatin domains, using false colors or quantitative microspectrophotometric analysis (Fig. 2a,b). Chromatin packaging, as induced by cell death and the action of oncogenes, carcinogens and hormones (Fig. 2c,d), or unraveling, as induced by drugs (Fig. 2e,f), can be assessed using this reaction (Vidal, 1984; Camby et al., 1995; Mello et al., 1995, Mello et al., 2007a,b; Vidal et al., 1998, 2014; Aldrovani et al., 2006; Mello, 2007; Felisbino et al., 2011; Poplineau et al., 2013; Veronezi et al., 2017). Feulgen-DNA image analysis has also been indicated for genome size measurements in a variety of animal and plant organisms (Hardie et al., 2002; Andraszek et al., 2009; Praça-Fontes et al., 2011; Bytyutskyy et al., 2012; Jeffery 2012 − among several reports). The results obtained with Feulgen-DNA image analysis have been shown to be compatible with those obtained by pulsed-field gel electrophoresis (Rincones et al., 2003). Due to changes in the morphological aspects of Feulgen-stained
Corresponding author. E-mail address: [email protected] (M.L.S. Mello). These authors share senior authorship.
http://dx.doi.org/10.1016/j.acthis.2017.07.002 Received 13 May 2017; Received in revised form 7 July 2017; Accepted 7 July 2017 0065-1281/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Mello, M.L.S., Acta Histochemica (2017), http://dx.doi.org/10.1016/j.acthis.2017.07.002
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Fig. 1. Ordinary (a–c) and fluorescence (d) microscopy views of Feulgen-stained chromatin. a. Mouse hepatocytes. b. Caco intestinal cells. The arrow indicates apoptotic bodies. c. and d. Malpighian tubule cell nuclei of Triatoma infestans where heterochromatin bodies are indicated (arrows). Bars equal 20 μm.
maximally (curve peak or plateau). The Feulgen-DNA values for cell nuclei are obtained by multiplying their absorbance values by the nuclear absorbing area. The DNA depurination phase is followed by a gradual breakdown and solubilization of the apurinic acid (descending branch of the hydrolysis curve) (Fig. 3). The kinetics of acid hydrolysis depends on the concentration, time and temperature of the acid bath (Feulgen and Rossenbeck, 1924; Sandritter et al., 1965; Andersson and Kjellstrand, 1975). Non-hydrolyzed DNA preparations will not develop a Feulgen-positive response. Depending on the hydrolysis conditions and the material under study, the breakdown of apurinic acid may attack a small portion of the DNA while it is still undergoing depurination (Savage and Plaut, 1958; Sandritter et al., 1965). To circumvent this phenomenon, the use of milder acid hydrolysis conditions (treatment with 3.5–4.0 M HCl at room temperature or 0.1 M HCl at 37 °C) than those originally proposed (1 M HCl at 60 °C) has been recommended (Tamm et al., 1952; Jobst, 1961; Sandritter et al., 1965). The simultaneous production and breakdown of apurinic acid aldehydes is previewed when fitting the Feulgen hydrolysis curves to the Bateman’s function (Böhm and Seibert, 1966; Mello, 1979). A previous knowledge or determination of the optimal hydrolysis time that corresponds to the peak or plateau of the Feulgen hydrolysis curve under specific hydrolysis conditions regarding
chromatin/chromosomes in association with varying cell functionality, the evaluation of mitotic indices, chromosome abnormalities, micronuclei frequency, apoptotic ratios and mitotic catastrophe ratios can also be performed in cell preparations subjected to this reaction (Camby et al., 1995; Unal et al., 2005; Mello, 2007; Felisbino et al., 2011, 2014; Veronezi et al., 2017). 2. Chemical principles The Feulgen reaction is comprised of two steps: 1. Acid hydrolysis, generally performed with an HCl solution; 2. Treatment with Schiff’s reagent, a leucoderivative from basic fuchsin. 2.1. Acid hydrolysis Acid hydrolysis denatures DNA and induces DNA depurination and deoxyribose residues unmasking, which makes them function as aldehydes. By gradually increasing hydrolysis times, DNA depurination will proceed (ascending branch of a Feulgen hydrolysis curve plotted with Feulgen-DNA values vs. hydrolysis times) until this event is reached 2
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Fig. 2. Differences in chromatin supraorganization revealed in Feulgen-stained cells. a. and b. Malpighian tubule cell nuclei of Triatoma infestans. Deeply condensed chromatin areas such as that of the heterochromatin body appear revealed in a different false color (orange). c. and d. An increased area covered with an orange false-color (d) in Feulgen-stained, benzo[a]pyrene-transformed human breast epithelial cells indicates increased chromatin condensation. e. and f. The Feulgenstained nuclei with decreased area covered with a green false-color (f) in valproic acid-treated HeLa cells indicate increased chromatin decondensation (reprinted from Felisbino et al., 2011 − PLoS ONE 6: e29144).
pararosaniline dye, in the presence of H2SO3 and water (for preparation, see Lillie, 1951 or the review by Chieco and Derenzini, 1999). The colorless characteristic of the Schiff reagent results from the binding of one H2SO3 molecule to the central carbon atom of pararosaniline, thereby changing its chromophoric structure. In the presence of aldehydes, which will bind the leucoderivative of the pararosaniline, the original chromophoric structure of this dye will be reestablished and consequently, a magenta color will be obtained. Two main theories have been proposed regarding the mechanisms of Schiff-aldehyde interactions. According to one hypothesis (Wieland and Scheuing, 1921), the Schiff reagent consists of a double sulfonated product (N-sulfinic acid). This product is generated because one H2SO3
the acid concentration and temperature is mandatory when the evaluation of the Feulgen-DNA content is required. 2.2. Schiff reagent treatment The second phase of the Feulgen reaction consists of the reaction of the DNA aldehydes that were exposed by DNA depurination with the aldehyde and ketone-specific Schiff reagent, which is originally colorless or straw-colored, resulting in a magenta-colored product. If aldehyde-blocking reagents are added to the preparations after hydrolysis, no reaction will occur with the Schiff’s reagent. The Schiff reagent is obtained from basic fuchsin, which is mostly composed of the 3
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et al., 1960; Gledhill, 1970). Although the analysis of these sperm cells using UV microspectrophotometry does not reveal differences in the DNA content compared to morphologically and chemically normal spermatozoa, an increase in the Feulgen staining intensity is demonstrated in the spermatozoa that contain a nuclear protein with an abnormal composition and that is bound weakly to the DNA. Consequently, these spermatozoa have a faster Feulgen depurination phase than chemically normal sperm cells and, as a result, they stain more intensely. Spermatozoa that exhibit more intense Feulgen staining during the depurination phase have been found to appear more frequently in subfertile bulls. An inverse phenomenon occurs during normal spermatogenesis, when spermatozoal nuclear basic proteins substitute for somatic histones (Gledhill, 1970). Sperm cells from different animal species that carry different types of basic protein in complex with DNA also differ in their depurination and apurinic acid breakdown kinetics (Silva and Mello, 1986). The same concept applies to erythrocytes from snakes of different species and ages (Miyamoto et al., 2005). The Feulgen hydrolysis kinetics of nuclear regions that contain significant amounts of heterochromatin or condensed chromatin may also differ in comparison to euchromatin or non-condensed chromatinrich regions (Mittermayer et al., 1971; Mello, 1979; Mello and Vidal, 1980). Different labilities to acid hydrolysis of DNA have been revealed in polytene chromosome regions that differ in their DNA composition and DNA-protein complexes (Mello and Vidal, 1980). Because different fixatives preserve proteins differently, the rate at which DNA responds to the hydrolytic action is affected by fixative solutions. Differences in acid hydrolysis curve profiles are therefore observed when cells fixed with a formalin-containing solution are compared to cells fixed in the absolute ethanol (or methanol)-acetic acid (3:1, v/v) solution that is usually employed for chromosome/ chromatin DNA studies (Mello and Vidal, 1978; Mello, 1979; Silva and Mello, 1986).
Fig. 3. Feulgen hydrolysis curve in which the ascending branch is due to gradual DNA depurination, the plateau represents maximal DNA depurination and the descending branch reveals the apurinic acid depolymerization.
molecule initially links to the central C atom of the pararosaniline, which leads to the loss of its chromophoric structure, and production of an intermediary compound (fuchsin leucosulfonic acid). Then, one SO2 group binds to one of the aromatic ammine radicals of leucosulfonic acid. With excess SO2, a second SO2 group can bind to the aromatic ammine radical vicinal to the first one (Kasten, 1960). The linking of the aldehyde groups of the apurinic acid to N-sulfinic acid occurs through a bridge between the C atoms of aldehydes and sulfonated groups of N-sulfinic acid. Then, the HSO3 radical bound to the central C atom is dislodged, which restores the central C double bond and the chromophoric structure to the product. According to an alternative theory, the Schiff reagent consists of bleached pararosaniline and excess SO2 (Prud’homme, 1900; Hörmann et al., 1958; Heinemann, 1970; Puchtler et al., 1975). The SO2 groups in an aqueous solution react first to apurinic acid aldehydes to form an alkyl-sulfonic acid. Then, the C atom of this acid links to the N atom of the primary aromatic ammine of the pararosaniline leucoderivative. This hypothesis is more likely because it is supported by the results of chromatography and electrophoresis assays of several reaction products derived from the Schiff reagent and formaldehyde (Barka and Ornstein, 1960; Hiraoka, 1960; Hardonk and van Duijn, 1964). For details on the formulas related to the proposed, above-mentioned mechanisms, the reviews by Puchtler et al. (1975) and Mello and Vidal (1978) are suggested. For the observation and quantification of Feulgen-stained DNA by fluorescence microscopy, dilution of the Schiff reagent has been recommended (Mello and Vidal, 1978; Burger et al., 1990; Alvarenga et al., 2011). RNA does not compete with DNA for Feulgen staining. Although RNA purines are also removed by acid hydrolysis, only deoxyriboses behave as true aldehydes that can react with the Schiff reagent; in addition, RNA is easily extracted by the acid hydrolysis step (Mello and Vidal, 1978).
3.2. Schiff reagent composition The basic fuchsin that is used to prepare the Schiff reagent should contain pure pararosaniline or be composed primarily of pararosaniline and traces of rosaniline and magenta II (which are all triphenylmethane dyes). The differences in the basic fuchsin composition depend on the trademark sources. Care should be taken to avoid contaminant dyes that may vary in type and content and that may affect the quantitative evaluation of the Feulgen staining (Cortelazzo et al., 1983). Although pararosaniline is conventionally used to prepare the Schiff reagent, other dyes, such as Doebner’s violet, diazofuchsin, neutral violet, pyronin B, and phenosafranine have also been proposed in several reports to compose Schiff-like reagents (Kasten, 1960). An osmium-ammine complex is used as a Schiff-like reagent for DNA staining in electron microscopy (Cogliati and Guatier, 1973; Mikhaylova and Markov, 1994; Chieco and Derenzini, 1999; Derenzini et al., 2014).
3. Factors that may affect the Feulgen-DNA response 3.3. Plasmal reaction 3.1. DNA availability for depurination and breakdown of apurinic acid Cells that contain cytoplasmic aldehydes may exhibit a false, nonspecific Feulgen-positive response (plasmal reaction) that could potentially affect the DNA analysis. This event has been verified in certain tissues even after fixation in acetic ethanol. The plasmal reaction has been reported to occur in Feulgen-stained, whole-mounted preparations of bee tissues in studies of aging effects and participation of DNA methylation in polyploid cells (Mampumbu et al., 2004; Peres et al., 2014). The “en bloc”, Feulgen-stained heart fragments used for studies of cardiomyocyte whole nuclei for detection of polyploidy and chromatin textural changes in normoglycemic aged and hyperglycemic adult mice also show signs of a plasmal reaction (Silva et al., 2013). To abolish this reaction, treatment of the preparations with a 5% sodium borohydride aqueous solution and an acetone-chloroform solution (1:1, v/v) for
In eukaryotic chromatin, DNA forms a complex with histones and non-histone proteins. The accessibility of DNA to depurination thus requires disruption of the linkages between the DNA and the proteins. Indeed, acid hydrolysis dissociates proteins from DNA; however, in some cases, a small amount of protein remains attached to the DNA. Both depurination and apurinic acid depolymerization depend on chromatin architecture and, consequently, on the hierarchical levels of the chromatin packing states. Nuclei with the same DNA content but containing different DNA-protein complexes may respond differently to the Feulgen reaction. A well-known example is the Feulgen response to DNA in morphologically normal bull spermatozoa bearing chemically abnormal nuclear proteins that are bound to the nucleic acid (Parez 4
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et al., 2005, 2007); 2) in benzo[a]pyrene-transformed human breast epithelial MCF-10F cells after microcell-mediated transfer of chromosomes (Mello et al., 2003); in MCF-10F cells transformed with 17-βestradiol via a non-estrogen α-receptor-mediated process and selected for aggressive invasiveness (Mello et al., 2007a,b); 4) in hepatocytes and cardiomyocytes from adult diabetic mice compared to aged normoglycemic mice, which highlighted dissimilarities between diabetes and aging processes (Ghiraldini et al., 2012; Silva et al., 2013); and in human hepatocellular carcinoma HepG2 cells cultivated under hyperglycemic conditions (Felisbino et al., 2016). Another form of assessing the DNA content in individual Feulgenstained cell nuclei is using an automatic scanning microspectrophotometry device. Development of specific software in association with hardware that adequately controls the automatic and predominantly unidirectional motion of scanning the preparations on the microscope stage and simultaneously estimating the absorbance at grid points of the stained material can enable researchers to detect specific image parameters. In the case of the Feulgen-stained preparations, these parameters may reveal Feulgen-DNA values and nuclear areas (AT and ST, respectively) as well as Feulgen-DNA values and areas for nuclear regions covered by a chromatin identified as presenting absorbances above a pre-selected cutoff point (“condensed” chromatin) (AC and SC, respectively). An average absorption ratio parameter (AAR = (AC/SC)/(AT/ST)) that defines the contrast between condensed and total chromatin can also be calculated (Vidal et al., 1973). A scatter diagram relating the percentage of nuclear areas covered by Feulgen-stained “condensed” chromatin (SC %) and the AAR parameter, has been proposed by Vidal (1984) using automatic scanning microspectrophotometry. The matching of the Sc% and AAR values defines points in the scatter diagram that correspond to individual nuclei with specific phenotypes (Fig. 4a,b). This diagram was useful for pattern recognition involving chromatin remodeling that occurs with aging (Vidal, 1984), fasting stress (Mello, 1989), cell transformation and tumorigenesis (Mello and Russo, 1990; Mello and Chambers, 1994; Mello et al., 1994; Mello et al., 1995; Mello et al., 2007a,b; Mello et al., 2009; Vidal et al., 1998), apoptosis (Mello, 2007) and drug effects (Felisbino et al., 2011, 2014; Vidal et al., 2014; Alvarenga et al., 2016). Although proposed for data obtained automatically in a system that uses a scanning microspectrophotometer, this plot can be perfectly constructed with values obtained using video image analysis (Mello et al., 1994, 1995). All of these features have contributed to our understanding of the changes of nuclear/chromatin supraorganization that are of interests in human, animal and plant cell biology, pathology, and genetics questions as demonstrated in several reports.
15 min each prior to the Feulgen staining is recommended (Mampumbu et al., 2004; Peres et al., 2014; Silva et al., 2013). 4. Feulgen absorption spectra Since the pioneering studies by Moses and Kasten (apud Mello and Vidal, 1978), the Feulgen spectral absorption curves obtained with increasing hydrolysis times have revealed not only an absorption peak at λ = 575–580 nm, but also a shoulder at λ = 530–550 nm. According to Deitch (1966), the main peak would be contributed by Schiff molecules that are bound to one apurinic acid aldehyde, whereas the absorption shoulder would be contributed by Schiff molecules that are bound to two vicinal aldehydes in the apurinic acid. With advancing hydrolysis times, there would be an increased opportunity for vicinal aldehydes to be exposed to and bind Schiff molecules (Deitch, 1966). This hypothesis gained support from an analysis of spectral absorption curves of the heterochromatin and euchromatin of Triatoma infestans nuclei subjected to an extended 0.1 M HCl mild hydrolysis (Mello, 1978). This heterochromatin contains a repetitive AT-rich DNA (Alvarenga et al., 2011), a fact that favored that the Schiff molecules became di-substituted with adjacent apurinic acid aldehydes 7 Å apart from each other. As expected, a well-prominent shoulder at λ ∼530 nm was detected in the Feulgen-stained heterochromatin of Triatoma infestans after a prolonged acid hydrolysis (Mello, 1978). The Feulgen reaction may thus provide information on the extent of vicinal purines in a chromatin region. 5. Evaluation of Feulgen-DNA content and other characteristics of Feulgen-stained nuclei The Feulgen-DNA content of individual nuclei can be measured and expressed as arbitrary units or picograms, depending on the reference the values are compared to. Feulgen-DNA classes named 1C, 2C, 4C, and so on, are associated with the DNA contents of haploid, diploid, tetraploid, and so on, nuclei of the same species and are employed in a large number of investigations. Ploidy intermediary classes can also be determined and are frequently associated with aneuploidy, DNA loss, polyploidy, and nuclear fusion. Other quantitative information on nuclear and chromatin image characteristics of the Feulgen-stained preparations can be described through assessment of geometric, densitometric and textural features using video cytometry combined with light or fluorescence microscopy and different software programs. Matching Feulgen-DNA values and any one of these features or matching two or more features for individual nuclei can be compared under different developmental stages or under various physiological or even pathological conditions of interest. Features, including entire nuclear areas and absorbing areas covered by chromatin; the nuclear perimeter, feret (a measure of the object axis along a specified direction), and shape (roundness); chromatin optical density (=absorbance) and variations on that density; a contrast between condensed and unpacked chromatin areas; chromatin entropy defined in terms of the number of bits required to store information; chromatin energy; level of peripheral distribution of condensed chromatin; and many other features contribute to defining specific nuclear phenotypes (Kontron Elektronic Imaging System, 1995; Oberholzer et al., 1996). Video image analysis with advanced userfriendly software in association with modern microscopic devices has been employed to increase the precision and reproducibility of diagnostic pathology (Marchevsky and Erler, 1994; Nielsen et al., 2012). In our laboratory, the assessment of the above-mentioned features, especially entropy and contrast between condensed and unpacked chromatin in Feulgen-stained preparations using video image cytometry, has allowed us to demonstrate global chromatin remodeling under specific experimental conditions. These changes were verified 1) in mouse hepatocytes under starvation (followed by full nourishment recovery after refeeding), development and aging conditions (Moraes
6. New perspectives of using of the Feulgen reaction for studies of chromatin texture related to epigenetics With current progress regarding the relationship between epigenetic marks, chromatin structure and functional implications, the Feulgen reaction has additional potential usefulness. In Feulgen-stained preparations, a significantly altered chromatin texture can be identified through observation of changes in false-color images or after performing a cytometric analysis. When used in association with the results obtained from other powerful methodologies, such as immunoassays, molecular biology tests, Raman microspectroscopy and Fourier transform-infrared microspectroscopy, an alteration in the higher-order organization of chromatin can effectively be related to global epigenetic changes (Felisbino et al., 2011, 2014; Poplineau et al., 2011; Rodrigues et al., 2014; Veronezi et al., 2017). Such a correlation can be exemplified better following the effects of the inhibition of histone deacetylases by drugs such as valproic acid (VPA) and trichostatin A (TSA) on chromatin. VPA, which is a drug widely prescribed for treatment of seizure disorders, is a potent inhibitor of class I histone deacetylases (Göttlicher et al., 2001; Phiel 5
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biology approaches. In conclusion, DNA characterization and quantification can still benefit from the Feulgen reaction, which remains pertinent for the purpose that it was originally developed for. Certainly, the Feulgen reaction, as with many other cytochemical procedures, is not competitive with current molecular biology assays. However, it can provide researchers with results that could have substantial value when combined with information obtained using modern biochemical and molecular biology techniques as well as advanced microscopy. The success of the use of the Feulgen reaction in new applications will depend on its choice to respond to innovative questions. The words of Santiago Ramón y Cajal may apply regarding the use of this method in new approaches: “Puede afirmarse que no hay cuestiones agotadas, sino hombres agotados en las cuestiones” (Translated as, “There are no exhausted matters but men exhausted in their questions”). Acknowledgments This study was sponsored by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant no. 2015/10356-2) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant no. 304668/2014-1). We thank Mr. Eli H.M. dos Anjos for the assistance with formatting Figs. 1, 2 and 4. References Aldrovani, M., Mello, M.L.S., Guaraldo, A.M.A., Vidal, B.C., 2006. Nuclear phenotypes and DNA fragmentation in tendon fibroblasts of NOD mice. Caryologia 59, 116–124. Alvarenga, E.M., Mondin, M., Martins, J.A., Rodrigues, V.L.C.C., Vidal, B.C., et al., 2011. Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug. Micron 42, 568–578. Alvarenga, E.M., RodPlease delete one d.drigues, V.L.C.C., Moraes, A.S., Naves, L.S., Mondin, M., Felisbino, M.B., Mello, M.L.S., 2016. Histone epigenetic marks in heterochromatin and euchromatin of the Chagas’ disease vector, Triatoma infestans. Acta Histochem. 118, 401–412. Andersson, G.K.A., Kjellstrand, P.T.T., 1975. A study of DNA depolymerisation during Feulgen acid hydrolysis. Histochem 43, 123–130. Andraszek, K., Wojcik, E., Gruzewska, A., Smalec, E., 2009. Genome size of the European domestic goose (Anser anser domesticus). Can. J. Animal Sci. 89, 449–455. Böhm, N., Seibert, H.U., 1966. Zur Bestimmung der Parameter der Bateman-Funktion bei der Auswertung von Feulgen-Hydrolysekurven. Histochemie 6, 260–266. Barka, T., Ornstein, L., 1960. Some observations on the reaction of Schiff reagent with aldehydes. J. Histochem. Cytochem. 8, 208–213. Biesterfeld, S., Beckers, S., Villa Cadenas, M.C., Schramm, M., 2011. Feulgen staining remains the gold standard for precise DNA image cytometry. Anticancer Res. 31, 53–58. Burger, G., Oberholzer, M., Vooijs, G.P., 1990. Advances in Analytical Cellular Pathology. Excerpta Medica Amsterdam. Bytyutskyy, D., Srp, J., Flajshans, M., 2012. Use of Feulgen image analysis densitometry to study the effect of genome size on nuclear size in polyploidy sturgeons. J. Appl. Ichthyol. 28, 704–708. Camby, I., Salmon, I., Danguy, A., Pasteels, J.L., Kiss, R., 1995. The use of the digital cell image analysis of Feulgen-stained nuclei to detect apoptosis. Histochem. Cell Biol. 104, 407–414. Chieco, P., Derenzini, M., 1999. The feulgen reaction 75 years on. Histochem. Cell Biol. 111, 345–358. Cogliati, R., Guatier, A., 1973. Mise en evidence de l’AND et des polysaccharides a l’aide d’un nouveau réactif de type. Schiff. C.R. Acad. Sci. (Paris) Ser. D 276, 3041–3044. Cortelazzo, A.L., Vidal, B.C., Mello, M.L.S., 1983. Basic fuchsins and the Schiff-aldehyde reaction. I. Spectral absorption characteristics in solution. Acta Histochem. 73, 121–133. Deitch, A.D., 1966. Cytophotometry of Nucleic Acids. In Introduction to Quantitative Cytochemistry. In: Wied, G.L. (Ed.), Acad. Press, New York and London, pp. 327–354. Demirel, D., Akyurek, N., Ramzy, I., 2013. Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions. Cytopathology 24, 105–112. Derenzini, M., Olins, A.L., Olins, D.E., 2014. Chromatin structure in situ: the contribution of DNA ultrastructural cytochemistry. Eur. J. Histochem. 58, e2307. Detich, N., Bovenzi, V., Szyf, M., 2003. Valproate induces replication-independent active DNA demethylation. J. Biol. Chem. 278, 27586–27592. Felisbino, M.B., Tamashiro, W.M.S.C., Mello, M.L.S., 2011. Chromatin remodeling, cell proliferation and cell death in valproic acid-treated HeLa cells. PLoS One 6, e29144. Felisbino, M.B., Gatti, M.S.V., Mello, M.L.S., 2014. Changes in chromatin structure in NIH 3T3 cells induced by valproic acid and trichostatin A. J. Cell Biochem. 115, 1937–1947. Felisbino, M.B., Alves da Costa, T., Gatti, M.S.V., Mello, M.L.S., 2016. Differential response of human hepatocyte chromatin to HDAC inhibitors as a function of microenvironmental glucose level. J. Cell Physiol. 231, 2257–2265. Feulgen, R., Rossenbeck, H., 1924. Mikroskopisch-chemischer Nachweis einer Nucleinsäure vom Typus des Thymonucleinsäure und die darauf beruhende elektive
Fig. 4. Scatter diagram representation of nuclear phenotypes studied in Feulgen-stained preparations. This plot relates SC% (nuclear areas covered with “condensed” chromatin) and AAR (textural contrast between “condensed” and overall chromatin). Each point in the diagram represents one nucleus with a specific phenotype. This scatter diagram was modified from Vidal (1984) and Vidal et al. (1998b) and is shown as a model (a) as well as a representation of the altered distribution of nuclear phenotypes of Feulgen-stained HeLa cells subjected to treatment with 0.05 mM VPA for 1 h (blue) compared to the untreated control (black) (b) (reprinted from Felisbino et al., 2011 − PLoS ONE 6: e29144).
et al., 2001). While inducing histone deacetylase inhibition, VPA induces a variable degree of increased acetylation in lysine 9 of histone H3 (H3K9ac) in several cell types, including HeLa, HepG2, Caco and non-transformed NIH 3T3 cells (Felisbino et al., 2011, 2014, 2016). In these cells, such a phenomenon is accompanied by dose-and time-dependent chromatin remodeling that could be revealed using image cytometry or scanning microspectrophotometry of Feulgen-stained preparations (Felisbino et al., 2011, 2014, 2016). Although the effect of VPA on histone acetylation is a rapid and transient process, the changes in chromatin higher-order packing states of the Feulgen-stained HeLa cells were sustained longer if the cells were cultivated in the absence of VPA after the drug treatment. This outcome raised the suspicion that in addition to inducing histone acetylation, VPA was affecting the cellular DNA methylation status. DNA demethylation is a long-lasting process that may affect specific genes or gene promoters of some tumor cell lines, which could cause epigenetic reprogramming (Detich et al., 2003; Marchion et al., 2005; Milutinovic et al., 2007; Xu et al., 2007; Kortenhorst et al., 2009; Felisbino et al., 2011, 2014; Veronezi et al., 2017). These effects accompany (or occur simultaneously to) chromatin unpack and changes in nuclear architecture. Using immunoassays for 5-methyl-cytosine and DNA infrared microspectroscopy, which identified the level of the contribution of eCH3 groups to the FT-IR spectra, the long-lasting chromatin loosening that was verified in the Feulgen-stained HeLa cells in response to VPA treatment could be associated with DNA demethylation (Veronezi et al., 2017). Thus, the Feulgen reaction is a promising approach for alerting on epigenetic changes that globally affect chromatin higher-order packing states and that could be studied in depth using specific molecular 6
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Nouvelles matières colorants à fonctions acides. Bull. Soc. Chim. 23, 456–457. Puchtler, H., Meloan, S.N., Brewton, B.R., 1975. On the history of basic fuchsin and aldehyde-Schiff reactions from 1862 to 1935. Histochemistry 41, 185–194. Rincones, J., Meinhardt, L.W., Vidal, B.C., Pereira, G.A.G., 2003. Electrophoretic karyotype analysis of Crinipellis perniciosa, the causal agent of witches’ broom disease of Theobroma cacao. Mycol. Res. 107, 452–458. Rodrigues, H.F., Souza, T.A., Ghiraldini, F.G., Mello, M.L.S., Moraes, A.S., 2014. Increased age is associated with epigenetic and structural changes in chromatin from neuronal nuclei. J. Cell Biochem. 115, 659–665. Sandritter, W., Jobst, K., Rakow, L., Bosselmann, K., 1965. Zur Kinetik der Feulgenreaktion bei verlängerter Hydrolysezeit: Cytophotometrische Messungen in sichtbaren und ultravioletten Licht. Histochemie 4, 420–437. Savage, R.E., Plaut, W., 1958. The effect of HCl hydrolysis on the retention of thymidine in DNA. J. Biophys. Biochem. Cytol. 4, 701–706. Silva, M.J.P., Mello, M.L.S., 1986. Lability to acid hydrolysis in some different DNAprotein complexes of spermatozoa. Acta Histochem. 78, 197–215. Silva, I.S., Ghiraldini, F.G., Mello, M.L.S., 2013. DNA content, ploidy degrees and nuclear phenotypes in adult diabetic and aged normoglycemic mouse cardiomyocytes. Mol. Biol. Cell 24, 1161–1162. Swift, H., 1953. The deoxyribose nucleic acid content of animal nuclei. Physiol. Zool. 23, 169–198. Swift, H., 1955. Quantitative aspects of nuclear nucleoproteins. Int. Rev. Cytol. 2, 1–76. Tamm, C., Hodes, M.E., Chargaff, E., 1952. The formation of apurinic acid from the deoxyribonucleic acid of calf thymus. J. Biol. Chem. 195, 49–63. Unal, M., Celik, A., Ates, N.A., Micozkadioglu, D., Derici, E., et al., 2005. Cytogenetic biomonitoring in children with chronic tonsillitis: micronucleus frequency in exfoliated buccal epithelium cells. Int. J. Pediatr. Otorhinol. 69, 1483–1488. Veronezi, G.M.B., Felisbino, M.B., Gatti, M.S.V., Mello, M.L.S., Vidal, B.C., 2017. DNA methylation changes in valproic acid-treated HeLa cells as assessed by image analysis, immunofluorescence and vibrational microspectroscopy. PLoS One 12, e0170740. Vidal, B.C., Schlüter, G., Moore, G.W., 1973. Cell nucleus pattern recognition: influence of staining. Acta Cytol. 17, 510–521. Vidal, B.C., Russo, J., Mello, M.L.S., 1998. DNA content and chromatin texture of benzo [a]pyrene-transformed human breast epithelial cells as assessed by image analysis. Exptl. Cell Res. 244, 77–82. Vidal, B.C., Felisbino, M.B., Mello, M.L.S., et al., 2014. Image analysis of chromatin remodelling. In functional analysis of DNA and chromatin. In: In: Stockert, J.C. (Ed.), Methods in Molecular Biology, vol. 1094. Springer Sci.-Humana Press, New York, pp. 99–108. Vidal, B.C., 1984. Polyploidy and nuclear phenotypes in salivary glands of the rat. Biol. Cell 50, 137–146. Wieland, H., Scheuing, G., 1921. 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Färbung von Zellkernen in mikroskopischen Präparaten. Hoppe-Seylers Z. Physiol. Chem. 135, 203–248. Göttlicher, M., Minucci, S., Zhu, P., Kramer, O.H., Schimpf, A., Giavara, S., Sleeman, J.P., Lo Coco, F., Nervi, C., Pelicci, P.G., Heinzel, T., 2001. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 20, 6969–6978. Ghiraldini, F.G., Silva, I.S., Mello, M.L.S., 2012. Polyploidy and chromatin remodeling in hepatocytes from insulin-dependent diabetic and normoglycemic aged mice. Cytometry Part A 81, 755–764. Gledhill, B.L., 1970. In: In: Wied, G.L. (Ed.), Changes in Nuclear Stainability Associated with Spermateliosis, Spermatozoal Maturation, and Male Infertility. In Introduction to Quantitative Cytochemistry, vol II. Acad Press, New York and London, pp. 125–151. Hörmann, H., Grassmann, W., Fries, G., 1958. Ueber den Mechanismus der Schiffschen Reaktion. Justus Liebigs Ann. Chem. 616, 125–147. Hardie, D.C., Gregory, T.R., Hebert, P.D.N., 2002. From pixels to pictograms: a begginners’ guide to genome quantification by Feulgen image analysis densitometry. J. Histochem. Cytochem. 50, 735–749. Hardonk, M.J., van Duijn, P., 1964. The mechanism of the Schiff reaction as studied with histochemical model systems. J. Histochem. Cytochem. 12, 748–751. Heinemann, R.L., 1970. Comparative spectroscopy of basic fuchsin, Schiff reagent, and a formalin-Schiff derivative in relation to dye structure and staining. Stain Technol. 45, 165–172. Hiraoka, T., 1960. Electrophoretic analysis of coloured products produced by Schiff-formaldehyde reaction. J. Biophys. Biochem. Cytol. 8, 286–288. Jeffery, N.W., 2012. The first genome size estimates for six species of krill (Malacostraca, Euphausiidae): large genomes at the north and south poles. Polar Biol. 35, 959–962. Jobst, K., 1961. Polarisationsoptische beobachtungen über die beziehungen der feulgenschen reaktion zur apurinsäure. Acta Histochem. 11, 222–229. Kasten, F.H., 1960. The chemistry of Schiff’s reagent. Int. Rev. Cytol. 10, 1–100. Kasten, F.H., 2003. Robert Feulgen and his histochemical reaction for DNA. Biotechn. Histochem. 78, 45–49. Kontron Elektronic Imaging System KS 400, 1995. User’s Guide Vol.1 (Eching/Munich). Kortenhorst, M.S.Q., Isharwal, S., van Diest, P., Chowdury, W.H., Marlow, C., et al., 2009. Valproic acid causes dose- and time-dependent changes in nuclear structure in prostate cancer cells in vitro and in vivo. Mol. Cancer Ther. 8, 802–808. Lillie, R.D., 1951. Simplification of the manufacture of Schiff reagent for use in histochemical procedures. Stain Techn. 26, 163–165. Mampumbu, A.R., Vidal, B.C., Mello, M.L.S., 2004. Feulgen staining in Malpighian tubules of meliponid bees: a methodological contribution. Braz. J. Morph. Sci. 21, 31–33. Marchevsky, A.M., Erler, B.S., 1994. Morphometry in pathology. In: Marchevsky, A.M., Bartels, P.H. (Eds.), Image Analysis: A Primer for Pathologists. Raven Press Ltd, New York, pp. 125–179. Marchion, D.C., Bicaku, E., Daud, A.I., Sullivan, D.M., Munster, P.N., 2005. Valproic acid alters chromatin structure by regulation of chromatin modulation proteins. Cancer Res. 65, 3815–3822. Mello, M.L.S., Chambers, A.F., 1994. Image analysis of Feulgen-stained transformed NIH 3T3 cells differing in p21 expression and ras-induced metastatic ability. Anal. Quant. Cytol. Histol. 16, 113–123. Mello, M.L.S., Russo, J., 1990. Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells. Biochem. Cell Biol. 68, 1026–1031. Mello, M.L.S., Vidal, B.C., 1978. A reação de Feulgen. Ciênc. Cult. 30, 665–676. Mello, M.L.S., Vidal, B.C., 1980. Acid lability of deoxyribonucleic acids of some polytene chromosome regions of Rhynchosciara americana. Chromosoma 81, 419–429. Mello, M.L.S., Vidal, B.C., Planding, W., Schenck, U., 1994. Image analysis: video system adequacy for the assortment of nuclear phenotypes based on chromatin texture evaluation. Acta Histochem. Cytochem. 27, 23–31. Mello, M.L.S., Contente, S., Vidal, B.C., Planding, W., Schenck, U., 1995. Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis. Expt. Cell Res. 220, 374–382. Mello, M.L.S., Vidal, B.C., Lareef, M.H., Hu, Y.F., Yang, X., Russo, J., 2003. DNA content, chromatin texture and nuclear morphology in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17. Cytometry Part A 52, 70–76. Mello, M.L.S., Russo, P., Russo, J., Vidal, B.C., 2007a. 17-β-estradiol affects nuclear image properties in MCF-10F human breast epithelial cells with tumorigenesis. Oncol. Rep. 18, 1475–1481. Mello, M.L.S., Vidal, B.C., Russo, I.H., Lareef, M.H., Russo, J., 2007b. DNA content and chromatin texture of human breast epithelial cells transformed with 17-β-estradiol and the estrogen antagonist ICI 182,780 as assessed by image analysis. Mut. Res. 617, 1–7. Mello, M.L.S., Russo, P., Russo, J., Vidal, B.C., 2009. Entropy of Feulgen-stained 17-βestradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis. Oncol. Rep. 21, 1483–1487. Mello, M.L.S., 1978. Feulgen-DNA absorption spectrum of euchromatin and constitutive heterochromatin. J. Histochem. Cytochem. 26, 1082–1086. Mello, M.L.S., 1979. Patterns of lability towards acid hydrolysis in heterochromatin and euchromatin of Triatoma infestans Klug. Cell. Mol. Biol. 24, 1–16. Mello, M.L.S., 1989. Nuclear fusion and change in chromatin packing state in response to starvation in Triatoma infestans. Revta. Brasil. Genét. 12, 485–498. Mello, M.L.S., 2007. Discrimination of Apoptotic Cells by Image Analysis. In New Developments in Cell Apoptosis Research. In: Corvin, A.J. (Ed.), Nova Sci. Publ. Inc., New York, pp. 273–287. Mikhaylova, V.T., Markov, D.V., 1994. An alternative method for preparation of Schifflike reagent from osmium-ammine complex for selective staining of DNA on thin lowicryl sections. J. Histochem. Cytochem. 42, 1643–1649.
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Review
The Feulgen reaction: A brief review and new perspectives Maria Luiza S. Mello
⁎,1
, Benedicto de Campos Vidal1
Department of Structural and Functional Biology, Institute of Biology, University of Campinas (Unicamp), 13083-862 Campinas, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Feulgen reaction DNA Cytochemistry Image analysis Epigenetics
The Feulgen reaction has been proposed by Robert Feulgen and Heinrich Rossenbeck for the identification of DNA nearly a hundred years ago. Since then, many other applications of this cytochemical/topochemical procedure at qualitative and quantitative level have been proposed in relation to DNA and its role in chromatin in human, animal and plant cells. In this article, we briefly review some fundamental aspects of the Feulgen reaction and current applications of such a method in studies of altered chromatin texture, including its association with or preceding changes in transcriptional activities and effect on epigenetic marks. Further perspectives on the use of the Feulgen reaction will depend of the proposal of innovative biological questions in which its reveals appropriate.
1. Basic proposals Feulgen reaction is a classic cytochemical/topochemical method that has allowed investigators to reveal DNA as a component of chromatin and chromosomes. Feulgen-positive images exhibit a magenta (red-purple) color when observed under ordinary light microscopy or a red color under fluorescence microscopy (Fig. 1a–d). Proposed nearly a hundred years ago by Robert Feulgen and Heinrich Rossenbeck (1924), the Feulgen reaction continues to offer an opportunity for many research insights in fields such as cytogenetics, cell biology and analytical cytopathology (Hardie et al., 2002; Biesterfeld et al., 2011; Nielsen et al., 2012), as well as in current chromatin remodeling studies following changes in transcriptional activities (Felisbino et al., 2011, 2014; Natarajan et al., 2012; Poplineau et al., 2013; Vidal et al., 2014; Veronezi et al., 2017). A historical perspective of Robert Feulgen’s life and the development of the cytochemical reaction that was named after him were reported by Kasten (2003). One of the first applications of the Feulgen reaction allowed Alfert, Lison and Pasteels to demonstrate that DNA replication occurs during the S phase of the cell cycle. The constancy of the average DNA content per haploid chromosome set in numerous animal and plant species and the establishment of polyploidy and aneuploidy in particular cell types could also be determined (Swift, 1953; Swift, 1955 − reviews; Chieco and Derenzini, 1999 − review; Hardie et al., 2002). The Feulgen reaction permits the analysis of DNA to occur specifically and stoichiometrically in individual cells using different methods of microspectrophotometric analysis and digital image analysis. It has
⁎
1
been considered an objective method with high interobserver reproducibility and a useful tool for diagnostic and prognostic purposes (Biesterfeld et al., 2011; Nielsen et al., 2012; Demirel et al., 2013; Poplineau et al., 2013). It even presents some advantages over flow cytometry, such as possible long-term storage of the biological material, visual control for semi-interactive recognition during the analysis, applicability to single cells and feasible determination of very small amounts of DNA (Praça-Fontes et al., 2011). In image analysis studies of chromatin supraorganization, the Feulgen reaction can identify nuclear phenotypes that discriminate regions corresponding to heterochromatin and euchromatin domains, using false colors or quantitative microspectrophotometric analysis (Fig. 2a,b). Chromatin packaging, as induced by cell death and the action of oncogenes, carcinogens and hormones (Fig. 2c,d), or unraveling, as induced by drugs (Fig. 2e,f), can be assessed using this reaction (Vidal, 1984; Camby et al., 1995; Mello et al., 1995, Mello et al., 2007a,b; Vidal et al., 1998, 2014; Aldrovani et al., 2006; Mello, 2007; Felisbino et al., 2011; Poplineau et al., 2013; Veronezi et al., 2017). Feulgen-DNA image analysis has also been indicated for genome size measurements in a variety of animal and plant organisms (Hardie et al., 2002; Andraszek et al., 2009; Praça-Fontes et al., 2011; Bytyutskyy et al., 2012; Jeffery 2012 − among several reports). The results obtained with Feulgen-DNA image analysis have been shown to be compatible with those obtained by pulsed-field gel electrophoresis (Rincones et al., 2003). Due to changes in the morphological aspects of Feulgen-stained
Corresponding author. E-mail address: [email protected] (M.L.S. Mello). These authors share senior authorship.
http://dx.doi.org/10.1016/j.acthis.2017.07.002 Received 13 May 2017; Received in revised form 7 July 2017; Accepted 7 July 2017 0065-1281/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Mello, M.L.S., Acta Histochemica (2017), http://dx.doi.org/10.1016/j.acthis.2017.07.002
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Fig. 1. Ordinary (a–c) and fluorescence (d) microscopy views of Feulgen-stained chromatin. a. Mouse hepatocytes. b. Caco intestinal cells. The arrow indicates apoptotic bodies. c. and d. Malpighian tubule cell nuclei of Triatoma infestans where heterochromatin bodies are indicated (arrows). Bars equal 20 μm.
maximally (curve peak or plateau). The Feulgen-DNA values for cell nuclei are obtained by multiplying their absorbance values by the nuclear absorbing area. The DNA depurination phase is followed by a gradual breakdown and solubilization of the apurinic acid (descending branch of the hydrolysis curve) (Fig. 3). The kinetics of acid hydrolysis depends on the concentration, time and temperature of the acid bath (Feulgen and Rossenbeck, 1924; Sandritter et al., 1965; Andersson and Kjellstrand, 1975). Non-hydrolyzed DNA preparations will not develop a Feulgen-positive response. Depending on the hydrolysis conditions and the material under study, the breakdown of apurinic acid may attack a small portion of the DNA while it is still undergoing depurination (Savage and Plaut, 1958; Sandritter et al., 1965). To circumvent this phenomenon, the use of milder acid hydrolysis conditions (treatment with 3.5–4.0 M HCl at room temperature or 0.1 M HCl at 37 °C) than those originally proposed (1 M HCl at 60 °C) has been recommended (Tamm et al., 1952; Jobst, 1961; Sandritter et al., 1965). The simultaneous production and breakdown of apurinic acid aldehydes is previewed when fitting the Feulgen hydrolysis curves to the Bateman’s function (Böhm and Seibert, 1966; Mello, 1979). A previous knowledge or determination of the optimal hydrolysis time that corresponds to the peak or plateau of the Feulgen hydrolysis curve under specific hydrolysis conditions regarding
chromatin/chromosomes in association with varying cell functionality, the evaluation of mitotic indices, chromosome abnormalities, micronuclei frequency, apoptotic ratios and mitotic catastrophe ratios can also be performed in cell preparations subjected to this reaction (Camby et al., 1995; Unal et al., 2005; Mello, 2007; Felisbino et al., 2011, 2014; Veronezi et al., 2017). 2. Chemical principles The Feulgen reaction is comprised of two steps: 1. Acid hydrolysis, generally performed with an HCl solution; 2. Treatment with Schiff’s reagent, a leucoderivative from basic fuchsin. 2.1. Acid hydrolysis Acid hydrolysis denatures DNA and induces DNA depurination and deoxyribose residues unmasking, which makes them function as aldehydes. By gradually increasing hydrolysis times, DNA depurination will proceed (ascending branch of a Feulgen hydrolysis curve plotted with Feulgen-DNA values vs. hydrolysis times) until this event is reached 2
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Fig. 2. Differences in chromatin supraorganization revealed in Feulgen-stained cells. a. and b. Malpighian tubule cell nuclei of Triatoma infestans. Deeply condensed chromatin areas such as that of the heterochromatin body appear revealed in a different false color (orange). c. and d. An increased area covered with an orange false-color (d) in Feulgen-stained, benzo[a]pyrene-transformed human breast epithelial cells indicates increased chromatin condensation. e. and f. The Feulgenstained nuclei with decreased area covered with a green false-color (f) in valproic acid-treated HeLa cells indicate increased chromatin decondensation (reprinted from Felisbino et al., 2011 − PLoS ONE 6: e29144).
pararosaniline dye, in the presence of H2SO3 and water (for preparation, see Lillie, 1951 or the review by Chieco and Derenzini, 1999). The colorless characteristic of the Schiff reagent results from the binding of one H2SO3 molecule to the central carbon atom of pararosaniline, thereby changing its chromophoric structure. In the presence of aldehydes, which will bind the leucoderivative of the pararosaniline, the original chromophoric structure of this dye will be reestablished and consequently, a magenta color will be obtained. Two main theories have been proposed regarding the mechanisms of Schiff-aldehyde interactions. According to one hypothesis (Wieland and Scheuing, 1921), the Schiff reagent consists of a double sulfonated product (N-sulfinic acid). This product is generated because one H2SO3
the acid concentration and temperature is mandatory when the evaluation of the Feulgen-DNA content is required. 2.2. Schiff reagent treatment The second phase of the Feulgen reaction consists of the reaction of the DNA aldehydes that were exposed by DNA depurination with the aldehyde and ketone-specific Schiff reagent, which is originally colorless or straw-colored, resulting in a magenta-colored product. If aldehyde-blocking reagents are added to the preparations after hydrolysis, no reaction will occur with the Schiff’s reagent. The Schiff reagent is obtained from basic fuchsin, which is mostly composed of the 3
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et al., 1960; Gledhill, 1970). Although the analysis of these sperm cells using UV microspectrophotometry does not reveal differences in the DNA content compared to morphologically and chemically normal spermatozoa, an increase in the Feulgen staining intensity is demonstrated in the spermatozoa that contain a nuclear protein with an abnormal composition and that is bound weakly to the DNA. Consequently, these spermatozoa have a faster Feulgen depurination phase than chemically normal sperm cells and, as a result, they stain more intensely. Spermatozoa that exhibit more intense Feulgen staining during the depurination phase have been found to appear more frequently in subfertile bulls. An inverse phenomenon occurs during normal spermatogenesis, when spermatozoal nuclear basic proteins substitute for somatic histones (Gledhill, 1970). Sperm cells from different animal species that carry different types of basic protein in complex with DNA also differ in their depurination and apurinic acid breakdown kinetics (Silva and Mello, 1986). The same concept applies to erythrocytes from snakes of different species and ages (Miyamoto et al., 2005). The Feulgen hydrolysis kinetics of nuclear regions that contain significant amounts of heterochromatin or condensed chromatin may also differ in comparison to euchromatin or non-condensed chromatinrich regions (Mittermayer et al., 1971; Mello, 1979; Mello and Vidal, 1980). Different labilities to acid hydrolysis of DNA have been revealed in polytene chromosome regions that differ in their DNA composition and DNA-protein complexes (Mello and Vidal, 1980). Because different fixatives preserve proteins differently, the rate at which DNA responds to the hydrolytic action is affected by fixative solutions. Differences in acid hydrolysis curve profiles are therefore observed when cells fixed with a formalin-containing solution are compared to cells fixed in the absolute ethanol (or methanol)-acetic acid (3:1, v/v) solution that is usually employed for chromosome/ chromatin DNA studies (Mello and Vidal, 1978; Mello, 1979; Silva and Mello, 1986).
Fig. 3. Feulgen hydrolysis curve in which the ascending branch is due to gradual DNA depurination, the plateau represents maximal DNA depurination and the descending branch reveals the apurinic acid depolymerization.
molecule initially links to the central C atom of the pararosaniline, which leads to the loss of its chromophoric structure, and production of an intermediary compound (fuchsin leucosulfonic acid). Then, one SO2 group binds to one of the aromatic ammine radicals of leucosulfonic acid. With excess SO2, a second SO2 group can bind to the aromatic ammine radical vicinal to the first one (Kasten, 1960). The linking of the aldehyde groups of the apurinic acid to N-sulfinic acid occurs through a bridge between the C atoms of aldehydes and sulfonated groups of N-sulfinic acid. Then, the HSO3 radical bound to the central C atom is dislodged, which restores the central C double bond and the chromophoric structure to the product. According to an alternative theory, the Schiff reagent consists of bleached pararosaniline and excess SO2 (Prud’homme, 1900; Hörmann et al., 1958; Heinemann, 1970; Puchtler et al., 1975). The SO2 groups in an aqueous solution react first to apurinic acid aldehydes to form an alkyl-sulfonic acid. Then, the C atom of this acid links to the N atom of the primary aromatic ammine of the pararosaniline leucoderivative. This hypothesis is more likely because it is supported by the results of chromatography and electrophoresis assays of several reaction products derived from the Schiff reagent and formaldehyde (Barka and Ornstein, 1960; Hiraoka, 1960; Hardonk and van Duijn, 1964). For details on the formulas related to the proposed, above-mentioned mechanisms, the reviews by Puchtler et al. (1975) and Mello and Vidal (1978) are suggested. For the observation and quantification of Feulgen-stained DNA by fluorescence microscopy, dilution of the Schiff reagent has been recommended (Mello and Vidal, 1978; Burger et al., 1990; Alvarenga et al., 2011). RNA does not compete with DNA for Feulgen staining. Although RNA purines are also removed by acid hydrolysis, only deoxyriboses behave as true aldehydes that can react with the Schiff reagent; in addition, RNA is easily extracted by the acid hydrolysis step (Mello and Vidal, 1978).
3.2. Schiff reagent composition The basic fuchsin that is used to prepare the Schiff reagent should contain pure pararosaniline or be composed primarily of pararosaniline and traces of rosaniline and magenta II (which are all triphenylmethane dyes). The differences in the basic fuchsin composition depend on the trademark sources. Care should be taken to avoid contaminant dyes that may vary in type and content and that may affect the quantitative evaluation of the Feulgen staining (Cortelazzo et al., 1983). Although pararosaniline is conventionally used to prepare the Schiff reagent, other dyes, such as Doebner’s violet, diazofuchsin, neutral violet, pyronin B, and phenosafranine have also been proposed in several reports to compose Schiff-like reagents (Kasten, 1960). An osmium-ammine complex is used as a Schiff-like reagent for DNA staining in electron microscopy (Cogliati and Guatier, 1973; Mikhaylova and Markov, 1994; Chieco and Derenzini, 1999; Derenzini et al., 2014).
3. Factors that may affect the Feulgen-DNA response 3.3. Plasmal reaction 3.1. DNA availability for depurination and breakdown of apurinic acid Cells that contain cytoplasmic aldehydes may exhibit a false, nonspecific Feulgen-positive response (plasmal reaction) that could potentially affect the DNA analysis. This event has been verified in certain tissues even after fixation in acetic ethanol. The plasmal reaction has been reported to occur in Feulgen-stained, whole-mounted preparations of bee tissues in studies of aging effects and participation of DNA methylation in polyploid cells (Mampumbu et al., 2004; Peres et al., 2014). The “en bloc”, Feulgen-stained heart fragments used for studies of cardiomyocyte whole nuclei for detection of polyploidy and chromatin textural changes in normoglycemic aged and hyperglycemic adult mice also show signs of a plasmal reaction (Silva et al., 2013). To abolish this reaction, treatment of the preparations with a 5% sodium borohydride aqueous solution and an acetone-chloroform solution (1:1, v/v) for
In eukaryotic chromatin, DNA forms a complex with histones and non-histone proteins. The accessibility of DNA to depurination thus requires disruption of the linkages between the DNA and the proteins. Indeed, acid hydrolysis dissociates proteins from DNA; however, in some cases, a small amount of protein remains attached to the DNA. Both depurination and apurinic acid depolymerization depend on chromatin architecture and, consequently, on the hierarchical levels of the chromatin packing states. Nuclei with the same DNA content but containing different DNA-protein complexes may respond differently to the Feulgen reaction. A well-known example is the Feulgen response to DNA in morphologically normal bull spermatozoa bearing chemically abnormal nuclear proteins that are bound to the nucleic acid (Parez 4
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et al., 2005, 2007); 2) in benzo[a]pyrene-transformed human breast epithelial MCF-10F cells after microcell-mediated transfer of chromosomes (Mello et al., 2003); in MCF-10F cells transformed with 17-βestradiol via a non-estrogen α-receptor-mediated process and selected for aggressive invasiveness (Mello et al., 2007a,b); 4) in hepatocytes and cardiomyocytes from adult diabetic mice compared to aged normoglycemic mice, which highlighted dissimilarities between diabetes and aging processes (Ghiraldini et al., 2012; Silva et al., 2013); and in human hepatocellular carcinoma HepG2 cells cultivated under hyperglycemic conditions (Felisbino et al., 2016). Another form of assessing the DNA content in individual Feulgenstained cell nuclei is using an automatic scanning microspectrophotometry device. Development of specific software in association with hardware that adequately controls the automatic and predominantly unidirectional motion of scanning the preparations on the microscope stage and simultaneously estimating the absorbance at grid points of the stained material can enable researchers to detect specific image parameters. In the case of the Feulgen-stained preparations, these parameters may reveal Feulgen-DNA values and nuclear areas (AT and ST, respectively) as well as Feulgen-DNA values and areas for nuclear regions covered by a chromatin identified as presenting absorbances above a pre-selected cutoff point (“condensed” chromatin) (AC and SC, respectively). An average absorption ratio parameter (AAR = (AC/SC)/(AT/ST)) that defines the contrast between condensed and total chromatin can also be calculated (Vidal et al., 1973). A scatter diagram relating the percentage of nuclear areas covered by Feulgen-stained “condensed” chromatin (SC %) and the AAR parameter, has been proposed by Vidal (1984) using automatic scanning microspectrophotometry. The matching of the Sc% and AAR values defines points in the scatter diagram that correspond to individual nuclei with specific phenotypes (Fig. 4a,b). This diagram was useful for pattern recognition involving chromatin remodeling that occurs with aging (Vidal, 1984), fasting stress (Mello, 1989), cell transformation and tumorigenesis (Mello and Russo, 1990; Mello and Chambers, 1994; Mello et al., 1994; Mello et al., 1995; Mello et al., 2007a,b; Mello et al., 2009; Vidal et al., 1998), apoptosis (Mello, 2007) and drug effects (Felisbino et al., 2011, 2014; Vidal et al., 2014; Alvarenga et al., 2016). Although proposed for data obtained automatically in a system that uses a scanning microspectrophotometer, this plot can be perfectly constructed with values obtained using video image analysis (Mello et al., 1994, 1995). All of these features have contributed to our understanding of the changes of nuclear/chromatin supraorganization that are of interests in human, animal and plant cell biology, pathology, and genetics questions as demonstrated in several reports.
15 min each prior to the Feulgen staining is recommended (Mampumbu et al., 2004; Peres et al., 2014; Silva et al., 2013). 4. Feulgen absorption spectra Since the pioneering studies by Moses and Kasten (apud Mello and Vidal, 1978), the Feulgen spectral absorption curves obtained with increasing hydrolysis times have revealed not only an absorption peak at λ = 575–580 nm, but also a shoulder at λ = 530–550 nm. According to Deitch (1966), the main peak would be contributed by Schiff molecules that are bound to one apurinic acid aldehyde, whereas the absorption shoulder would be contributed by Schiff molecules that are bound to two vicinal aldehydes in the apurinic acid. With advancing hydrolysis times, there would be an increased opportunity for vicinal aldehydes to be exposed to and bind Schiff molecules (Deitch, 1966). This hypothesis gained support from an analysis of spectral absorption curves of the heterochromatin and euchromatin of Triatoma infestans nuclei subjected to an extended 0.1 M HCl mild hydrolysis (Mello, 1978). This heterochromatin contains a repetitive AT-rich DNA (Alvarenga et al., 2011), a fact that favored that the Schiff molecules became di-substituted with adjacent apurinic acid aldehydes 7 Å apart from each other. As expected, a well-prominent shoulder at λ ∼530 nm was detected in the Feulgen-stained heterochromatin of Triatoma infestans after a prolonged acid hydrolysis (Mello, 1978). The Feulgen reaction may thus provide information on the extent of vicinal purines in a chromatin region. 5. Evaluation of Feulgen-DNA content and other characteristics of Feulgen-stained nuclei The Feulgen-DNA content of individual nuclei can be measured and expressed as arbitrary units or picograms, depending on the reference the values are compared to. Feulgen-DNA classes named 1C, 2C, 4C, and so on, are associated with the DNA contents of haploid, diploid, tetraploid, and so on, nuclei of the same species and are employed in a large number of investigations. Ploidy intermediary classes can also be determined and are frequently associated with aneuploidy, DNA loss, polyploidy, and nuclear fusion. Other quantitative information on nuclear and chromatin image characteristics of the Feulgen-stained preparations can be described through assessment of geometric, densitometric and textural features using video cytometry combined with light or fluorescence microscopy and different software programs. Matching Feulgen-DNA values and any one of these features or matching two or more features for individual nuclei can be compared under different developmental stages or under various physiological or even pathological conditions of interest. Features, including entire nuclear areas and absorbing areas covered by chromatin; the nuclear perimeter, feret (a measure of the object axis along a specified direction), and shape (roundness); chromatin optical density (=absorbance) and variations on that density; a contrast between condensed and unpacked chromatin areas; chromatin entropy defined in terms of the number of bits required to store information; chromatin energy; level of peripheral distribution of condensed chromatin; and many other features contribute to defining specific nuclear phenotypes (Kontron Elektronic Imaging System, 1995; Oberholzer et al., 1996). Video image analysis with advanced userfriendly software in association with modern microscopic devices has been employed to increase the precision and reproducibility of diagnostic pathology (Marchevsky and Erler, 1994; Nielsen et al., 2012). In our laboratory, the assessment of the above-mentioned features, especially entropy and contrast between condensed and unpacked chromatin in Feulgen-stained preparations using video image cytometry, has allowed us to demonstrate global chromatin remodeling under specific experimental conditions. These changes were verified 1) in mouse hepatocytes under starvation (followed by full nourishment recovery after refeeding), development and aging conditions (Moraes
6. New perspectives of using of the Feulgen reaction for studies of chromatin texture related to epigenetics With current progress regarding the relationship between epigenetic marks, chromatin structure and functional implications, the Feulgen reaction has additional potential usefulness. In Feulgen-stained preparations, a significantly altered chromatin texture can be identified through observation of changes in false-color images or after performing a cytometric analysis. When used in association with the results obtained from other powerful methodologies, such as immunoassays, molecular biology tests, Raman microspectroscopy and Fourier transform-infrared microspectroscopy, an alteration in the higher-order organization of chromatin can effectively be related to global epigenetic changes (Felisbino et al., 2011, 2014; Poplineau et al., 2011; Rodrigues et al., 2014; Veronezi et al., 2017). Such a correlation can be exemplified better following the effects of the inhibition of histone deacetylases by drugs such as valproic acid (VPA) and trichostatin A (TSA) on chromatin. VPA, which is a drug widely prescribed for treatment of seizure disorders, is a potent inhibitor of class I histone deacetylases (Göttlicher et al., 2001; Phiel 5
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biology approaches. In conclusion, DNA characterization and quantification can still benefit from the Feulgen reaction, which remains pertinent for the purpose that it was originally developed for. Certainly, the Feulgen reaction, as with many other cytochemical procedures, is not competitive with current molecular biology assays. However, it can provide researchers with results that could have substantial value when combined with information obtained using modern biochemical and molecular biology techniques as well as advanced microscopy. The success of the use of the Feulgen reaction in new applications will depend on its choice to respond to innovative questions. The words of Santiago Ramón y Cajal may apply regarding the use of this method in new approaches: “Puede afirmarse que no hay cuestiones agotadas, sino hombres agotados en las cuestiones” (Translated as, “There are no exhausted matters but men exhausted in their questions”). Acknowledgments This study was sponsored by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant no. 2015/10356-2) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant no. 304668/2014-1). We thank Mr. Eli H.M. dos Anjos for the assistance with formatting Figs. 1, 2 and 4. References Aldrovani, M., Mello, M.L.S., Guaraldo, A.M.A., Vidal, B.C., 2006. Nuclear phenotypes and DNA fragmentation in tendon fibroblasts of NOD mice. Caryologia 59, 116–124. Alvarenga, E.M., Mondin, M., Martins, J.A., Rodrigues, V.L.C.C., Vidal, B.C., et al., 2011. Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug. Micron 42, 568–578. Alvarenga, E.M., RodPlease delete one d.drigues, V.L.C.C., Moraes, A.S., Naves, L.S., Mondin, M., Felisbino, M.B., Mello, M.L.S., 2016. Histone epigenetic marks in heterochromatin and euchromatin of the Chagas’ disease vector, Triatoma infestans. Acta Histochem. 118, 401–412. Andersson, G.K.A., Kjellstrand, P.T.T., 1975. A study of DNA depolymerisation during Feulgen acid hydrolysis. Histochem 43, 123–130. Andraszek, K., Wojcik, E., Gruzewska, A., Smalec, E., 2009. Genome size of the European domestic goose (Anser anser domesticus). Can. J. Animal Sci. 89, 449–455. Böhm, N., Seibert, H.U., 1966. Zur Bestimmung der Parameter der Bateman-Funktion bei der Auswertung von Feulgen-Hydrolysekurven. Histochemie 6, 260–266. Barka, T., Ornstein, L., 1960. Some observations on the reaction of Schiff reagent with aldehydes. J. Histochem. Cytochem. 8, 208–213. Biesterfeld, S., Beckers, S., Villa Cadenas, M.C., Schramm, M., 2011. Feulgen staining remains the gold standard for precise DNA image cytometry. Anticancer Res. 31, 53–58. Burger, G., Oberholzer, M., Vooijs, G.P., 1990. Advances in Analytical Cellular Pathology. Excerpta Medica Amsterdam. Bytyutskyy, D., Srp, J., Flajshans, M., 2012. Use of Feulgen image analysis densitometry to study the effect of genome size on nuclear size in polyploidy sturgeons. J. Appl. Ichthyol. 28, 704–708. Camby, I., Salmon, I., Danguy, A., Pasteels, J.L., Kiss, R., 1995. The use of the digital cell image analysis of Feulgen-stained nuclei to detect apoptosis. Histochem. Cell Biol. 104, 407–414. Chieco, P., Derenzini, M., 1999. The feulgen reaction 75 years on. Histochem. Cell Biol. 111, 345–358. Cogliati, R., Guatier, A., 1973. Mise en evidence de l’AND et des polysaccharides a l’aide d’un nouveau réactif de type. Schiff. C.R. Acad. Sci. (Paris) Ser. D 276, 3041–3044. Cortelazzo, A.L., Vidal, B.C., Mello, M.L.S., 1983. Basic fuchsins and the Schiff-aldehyde reaction. I. Spectral absorption characteristics in solution. Acta Histochem. 73, 121–133. Deitch, A.D., 1966. Cytophotometry of Nucleic Acids. In Introduction to Quantitative Cytochemistry. In: Wied, G.L. (Ed.), Acad. Press, New York and London, pp. 327–354. Demirel, D., Akyurek, N., Ramzy, I., 2013. Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions. Cytopathology 24, 105–112. Derenzini, M., Olins, A.L., Olins, D.E., 2014. Chromatin structure in situ: the contribution of DNA ultrastructural cytochemistry. Eur. J. Histochem. 58, e2307. Detich, N., Bovenzi, V., Szyf, M., 2003. Valproate induces replication-independent active DNA demethylation. J. Biol. Chem. 278, 27586–27592. Felisbino, M.B., Tamashiro, W.M.S.C., Mello, M.L.S., 2011. Chromatin remodeling, cell proliferation and cell death in valproic acid-treated HeLa cells. PLoS One 6, e29144. Felisbino, M.B., Gatti, M.S.V., Mello, M.L.S., 2014. Changes in chromatin structure in NIH 3T3 cells induced by valproic acid and trichostatin A. J. Cell Biochem. 115, 1937–1947. Felisbino, M.B., Alves da Costa, T., Gatti, M.S.V., Mello, M.L.S., 2016. Differential response of human hepatocyte chromatin to HDAC inhibitors as a function of microenvironmental glucose level. J. Cell Physiol. 231, 2257–2265. Feulgen, R., Rossenbeck, H., 1924. Mikroskopisch-chemischer Nachweis einer Nucleinsäure vom Typus des Thymonucleinsäure und die darauf beruhende elektive
Fig. 4. Scatter diagram representation of nuclear phenotypes studied in Feulgen-stained preparations. This plot relates SC% (nuclear areas covered with “condensed” chromatin) and AAR (textural contrast between “condensed” and overall chromatin). Each point in the diagram represents one nucleus with a specific phenotype. This scatter diagram was modified from Vidal (1984) and Vidal et al. (1998b) and is shown as a model (a) as well as a representation of the altered distribution of nuclear phenotypes of Feulgen-stained HeLa cells subjected to treatment with 0.05 mM VPA for 1 h (blue) compared to the untreated control (black) (b) (reprinted from Felisbino et al., 2011 − PLoS ONE 6: e29144).
et al., 2001). While inducing histone deacetylase inhibition, VPA induces a variable degree of increased acetylation in lysine 9 of histone H3 (H3K9ac) in several cell types, including HeLa, HepG2, Caco and non-transformed NIH 3T3 cells (Felisbino et al., 2011, 2014, 2016). In these cells, such a phenomenon is accompanied by dose-and time-dependent chromatin remodeling that could be revealed using image cytometry or scanning microspectrophotometry of Feulgen-stained preparations (Felisbino et al., 2011, 2014, 2016). Although the effect of VPA on histone acetylation is a rapid and transient process, the changes in chromatin higher-order packing states of the Feulgen-stained HeLa cells were sustained longer if the cells were cultivated in the absence of VPA after the drug treatment. This outcome raised the suspicion that in addition to inducing histone acetylation, VPA was affecting the cellular DNA methylation status. DNA demethylation is a long-lasting process that may affect specific genes or gene promoters of some tumor cell lines, which could cause epigenetic reprogramming (Detich et al., 2003; Marchion et al., 2005; Milutinovic et al., 2007; Xu et al., 2007; Kortenhorst et al., 2009; Felisbino et al., 2011, 2014; Veronezi et al., 2017). These effects accompany (or occur simultaneously to) chromatin unpack and changes in nuclear architecture. Using immunoassays for 5-methyl-cytosine and DNA infrared microspectroscopy, which identified the level of the contribution of eCH3 groups to the FT-IR spectra, the long-lasting chromatin loosening that was verified in the Feulgen-stained HeLa cells in response to VPA treatment could be associated with DNA demethylation (Veronezi et al., 2017). Thus, the Feulgen reaction is a promising approach for alerting on epigenetic changes that globally affect chromatin higher-order packing states and that could be studied in depth using specific molecular 6
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