Feulgen Hydrolysis Research Articles

Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes

In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions.

DNA content of tumours: Cytophotometric measurements

A short account of cytophotometric investigations on malignant and on early cancerous lesions is given. It was possible to show, that human tumours exhibit an atypical DNA distribution pattern (DNA stem line). The carcinoma in situ of the exocervix shows similar as in malignant tumours a DNA stem line. This phenomenon can be traced also in the stage of invasion. The atypical course of Feulgen hydrolysis in tumour cells is related to a differential sensivitity towards acids of eu- and heterochromatin.

Quantitative assessment of bladder carcinoma by acid labile DNA assay.

Effective noninvasive methods for monitoring patients with bladder carcinoma and screening for bladder carcinoma that show better performance than the methods currently in use would be desirable for detecting urothelial carcinoma at an early, easily treatable stage. A rapidly hydrolyzed component of nuclear DNA has been described, the increase of which has been linked to malignancy. Quantitative determination of acid labile DNA has been applied successfully to detect other neoplasms. This study investigates the potential of this method to detect transitional cell carcinomas. Touch imprints of transurethral resection material from 62 cases of nonmalignant urothelium (control group including reactive changes) and 94 cases of bladder carcinoma were analyzed. The full Feulgen hydrolysis profiles of the nonmalignant and malignant urothelial cells were compared by measuring the staining density of the nuclei using digital image analysis after various hydrolysis times. Twenty cells were sampled randomly from among the cells measured for calculation of the mean optical density (MOD). The MOD of each time was used to build the hydrolysis profile of a case. A hydrolysis time of 10 minutes was found to be the most discriminative between control and carcinoma cases. Applying a single threshold MOD value of 101 resulted in a test sensitivity of 95.7%, a specificity of 94.4%, a positive predictive value of 98.3%, a negative predictive value of 95.9%, and an area under the receiver operating characteristic curve of 0.97. The results of this pilot study suggest that measurement of the rapidly hydrolyzed component of DNA present in the nuclei of bladder urothelium offers a highly sensitive and reliable supplement to the qualitative and subjective cytologic procedures currently in use.

A Preliminary Ploidy Analysis of Diploid and Triploid Salmonids

The findings indicate that mosaicism does occur in triploids and especially in triploid hybrids. Immature diploid and triploid salmon were obtained from the University of Alaska – National Marine Fisheries Service. These were fixed in 10% buffered formalin and blocks taken from various tissues including kidney, liver, spleen, and gonads. The paraffin blocks were sectioned at 4 μm and stained with hematoxylin and eosin for histologic evaluation and to quantitate nuclear DNA. The slides for DNA evaluation were stained using the Cell Analysis Systems, Inc. (CAS, Elmhurst, IL) kit following Feulgen hydrolysis. The Cell Analysis Systems, Inc. (Model 200) computer-assisted image analyzer was used for DNA quantitation. An attempt was made to evaluate the frequency of mosaicism, ploidy variation in somatic and gonadal tissues of triploids and hybrids as compared with normal diploids. Hepatic ploidy variation, normal in adult animals, was not present in these specimens. However, in gonads there was evidence of increased mosaicism among the triploids. Because of mosaicism in gonadal tissue, sterility of triploids cannot be presumed with certainty at this stage. Further studies are planned to examine this and other questions raised by these results.

DNA and keratin analysis of oral exfoliative cytology in the detection of oral cancer

Refinements in oral exfoliative cytology may make it a suitable screening technique for the early diagnosis of oral cancer. In this study DNA range profiles were combined with keratin expression in an attempt to improve the diagnostic accuracy of oral exfoliative cytology. Smears were taken from 33 biopsy-proven oral cancers and the contralateral normal site. For DNA range profiles the smears underwent Feulgen hydrolysis, with DNA distribution being assessed using the Vickers M85 microdensitometer. For keratin expression a panel of antikeratin antibodies were applied. The smears for keratin expression were then graded on a three-point scale. Abnormal DNA range profiles were observed in 23 of 33 smears taken from oral cancers and in two smears from normal oral mucosa (sensitivity 70%, specificity 90%, positive predictive value 90%). The simple epithelial keratins 8 and 19 were identified in the majority of oral cancer smears. The sensitivity of keratin 19 was greater (90%). However, keratin 8 was the most useful keratin marker associated with malignancy (sensitivity 62%, specificity 100%, positive predictive value 100%). The combination of simple keratin expression and DNA content improved the cancer detection rate beyond that obtainable with DNA range profile alone.

Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells.

We tested a method of measuring DNA content (Feulgen) and tritiated thymidine ([3H]-T) and bromodeoxyuridine (BrdU) incorporation by the same cell. Initial experiments showed that Feulgen hydrolysis denatured the DNA of fixed cells sufficiently to allow detection of incorporated BrdU with monoclonal antibodies. MCa-11 cells were then double-labeled with [3H]-T and BrdU, placed on slides, and Feulgen stained. Next, absorption cytometry was performed to measure the DNA content of randomly selected cells. Feulgen staining and the development and removal of either the [3H]-T or the BrdU grains after DNA measurements did not interfere with subsequent detection of the grains from the other label, and BrdU and [3H]-T can be used reliably in combination for identification of S-phase cells. This method may eventually allow the use of microscope-based image analysis to selectively measure the DNA contents and the BrdU/[3H]-T labeling of non-transformed stromal and cancer cells in solid tumors, thereby providing new insights into the growth kinetics of these heterogeneous cell populations.

Detection of field change in oral cancer using oral exfoliative cytologic study

Four smears were taken from the normal buccal mucosa of 55 oral cancer patients and 76 cancer-free patients. In each case, two were stained by the Papanicolaou method and two underwent Feulgen hydrolysis. Quantitative assessment of nuclear area (NA) and cytoplasmic area (CA) of the Papanicolaou smears was undertaken using a semiautomatic image analysis system. DNA profiles were assessed from the Feulgen smears using a Vickers M85 microdensitometer (Vickers Instruments, York, England) and were found to be diploid for all patients. Results were then analyzed with respect to those patients who took alcohol, tobacco, combination of alcohol and tobacco, and those who took neither. A significant reduction in CA for the oral cancer group (P equals 0.001) but no change in NA (P equals 0.74) was observed. A detailed analysis of alcohol and tobacco habits could identify no significant role for these two factors, in the reduction in cytoplasmic area. Such field change may prove to be of value in predicting the development of second malignant tumors.

Quantitative determination of acid-labile dna in cervical intraepithelial neoplasia

Using a modified Feulgen hydrolysis procedure and integrating microdensitometry, the acid-labile nuclear DNA in exfoliated cervical epithelial cells was quantified in a range of histologically confirmed cervical intraepithelial neoplasia (CIN), invasive cancer, and normal controls. The mean relative optical densities obtained for each sample group showed an increase from normal epithelium, through CIN grades, to invasive cancer. Although there was some overlap between groups, the difference in the overall mean values between the adjacent groups was statistically significant. The sensitivity of the test was 87.1% with a specificity of 99.2% and a predictive value of 99.5%, with no false negatives in the severe dysplasia and cancer groups. Quantitative data allows the threshold value to be altered to vary the sensitivity and specificity according to prevailing requirements. This suggests the possibility of using quantitative acid-labile DNA measurements to improve existing screening for cervical precancer.

A histological and flow cytometric study of dog brain endothelial cell injuries in delayed radiation necrosis

The pathogenesis of delayed cerebral radiation necrosis was studied histologically and biochemically in 25 dogs with special attention to vascular endothelial cell injuries. The dogs were sacrificed 3 to 30 months after irradiation with a single dose of 15 Gy to the head. Brain specimens were appropriately fixed for light and electron microscopic studies, and capillary endothelial cells were isolated for flow cytometric study. The endothelial cells were stained with acridine orange, then the cell ratios in the reproductive phase (S + G2 + M) were investigated with flow cytometry. Thereafter, Feulgen hydrolysis and computer analysis of the hydrolysis curves were performed to examine the qualitative changes in deoxyribonucleic acid (DNA) of endothelial cells after irradiation. Under light microscopy, spongy degeneration with small cell infiltration was observed, especially in the frontal white matter, at 6 months after irradiation. At 9 months, necrotic foci appeared and developed until 15 months after irradiation. Blood vessels around the necrotic area showed luminal narrowing with endothelial hyperplasia and proliferation. At 30 months, no fresh necrotic lesions were observed. Under electron microscopy, endothelial cells of capillaries and small vessels around the necrotic area showed an increase of pinocytosis, and in the nuclei there was an increase of infoldings and euchromatin. The cell ratios in the reproductive phase were 14.5% to 23.3% (maximum at 9 months) in the irradiated group compared to 6.4% in the control group. The rate constant of apurinic acid production, a parameter correlating with DNA transcriptional activity, was minimum at 3 months and maximum at 9 months after irradiation. The data suggest that impairment of the microcirculation plays an important role in the pathogenesis of delayed radiation necrosis, and that the time of necrosis occurrence closely correlates with the cell cycle of vascular endothelial cells.

Application of Fluorescence Cytophotometry to Biological Specimens

Recent advances in theoretical and practical aspects of the application of fluorescence cytophotometry to biological specimens were described. A new method of quantitative detection of DNA damage by computer analysis of Feulgen hydrolysis curve was introduced. The rate constans of depurination and depolymerization in Feulgen hydrolysis can be expressed in the form of k=AN2·exp (-E/RT), where A is a constant, N is HCl concentration, E is the activation energy, R is the gas constant, and T is the absolute temperature. And the Feulgen hydrolysis curve is determined by y (t) =y0k1/ (k2-k1) · (ek1t-ek2t), where y (t) is the amount of apurinic acid stainable with Feulgen reaction at hydrolysis time t, y0 is the amount of apurinic acid theoretically present at t=0. Computer fitting of Feulgen hydrolysis curve to these function makes it possible to determine the values of y0, k1, k2, and E. 1/k1 correlates with the degree of chromatin condensation, k2 reflects the degree of DNA instability, and y0 corresponds to the yield of single-stranded DNA induced by DNA damage due to aging, chemical carcinogen, radiation etc. The values of y0 and k2 are larger for the nuclear DNA in cancerous cells than in noncancerous cells. The increased DNA instability in cancerous cells made it possible to differentially stain cancerous cells with acridine orange after acid hydrolysis. This new technique is a useful tool for detecting malignancy in exfoliative cytology and malignant transformation by chemical carcinogen treatment. Some applications of fluorescence cytophotometry on viable cells were also described.

Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B.

Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.

A Combined Stain for Identifying Epithelial Cells of the Gastric Mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.

Lability to acid hydrolysis in some different DNA-Protein complexes of spermatozoa

The Feulgen hydrolysis kinetics was investigated in spermatozoa with different composition in DNA-protein complexes. The species used were: Bos taurus (arginine rich nuclear protein also containing cystine residues), Pichroplus bergi, Triatoma infestans (arginine-rich nuclear protein), Lytechinus variegatus and Apis mellifera (lysine-rich nuclear protein). The spermatozoa were subjected to Feulgen's reaction, after varying the fixative type and the hydrolysis times. Feulgen-DNA values were obtained with an automatic scanning cytophotometric procedure. Differences were demonstrated in the hydrolysis kinetics as a function of differences in composition of the DNA-protein complexes being present in the spermatozoon chromatin. Differences in the profiles of Feulgen hydrolysis curves, having for basis the fixation, were rather clear for bull, grasshopper, and blood-sucking insect spermatozoa than for the sea-urchin and bee spermatozoa. The different hydrolysis kinetics of chromatin of blood-sucking insect spermatozoa compared to that of grasshopper, sea-urchin, and bee sperm cells suggests that, although the first 2 materials contain an arginine-rich "germinative" protein and the latter 2 ones contain a lysine-rich protein, these differ to each other. The DNA depurination was obtained more quickly for T. infestans (20 min) and P. bergi (10 min) spermatozoa when they were fixed in the ethanol-acetic acid (EA) mixture. Morphologically anomalous bull spermatozoa fixed in the EA mixture presented a quicker depurination (30 min) as compared to the normal cells (1 h). The fast lability to acid hydrolysis in the abnormal cells is certainly due to anomalies in their basic nuclear "germinative" protein. In the formalin fixed materials the maximal depurination was obtained earlier in bull spermatozoa (30 min) followed by sperm cells of P. bergi, T. infestans, L. variegatus (all of them one-hour hydrolysis) and finally Apis mellifera (2 h hydrolysis). The presence of secondary peaks at the descending branch of the hydrolysis curves of grasshopper and sea-urchin spermatozoa, indicates for these, more than 1 kind of apurinic-acid protein complexes. The spermatozoa bearing arginine-and/or cystine-rich nuclear protein contain a more easily soluble apurinic acid protein complex. Due to the differences in hydrolysis kinetics of chromatin in spermatozoa containing different nuclear "germinative" proteins, this cellular type does not appear indicated as a haploid control for evaluation of Feulgen-DNA contents of diploid and polyploid somatic cells.

Quantitative detection of DNA damage in the neuronal cells of the cerebellum and cerebrum by the analysis of Feulgen hydrolysis curves

Touch smears of the cerebellum and cerebrum of ageing rats were fixed with methanol, hydrolyzed with 2 N HCl at various temperatures and for various periods, and stained with pararosaniline-Schiff reagent. The hydrolysis curves were determined by fluorescence cytophotometry and were computer fitted to the Bateman function to determine the kinetic parameters, the initial yield of apurinic acid or single-stranded DNA (y0), and the rate constants for depurination or denaturation (k1) and depolymerization (k2). The values for k1 (1/k1 is correlated with the degree of chromatin condensation) and k2 (which reflects the degree of DNA instability) steadily increased with age. The values for y0, which may indicate the degree of DNA denaturation or damage present before acid hydrolysis, also increased with age in both the cerebellum and cerebrum; however, this value was lower in the cerebellum until 15 weeks, with the situation being reversed after 35 weeks, the cross-over time being at about 25 weeks. The values of lnk1 and lnk2 were plotted as the function of the reciprocal of the absolute temperature (T) (Arrhenius plot) for both the cerebellum and cerebrum of 15- and 74-week-old rats, and the activation energies (E) for depurination and depolymerization were calculated from the slopes. In particular, the values of E for k2 decreased much more quickly with age and were smaller in cerebellum. In conclusion, the degree of DNA damage and DNA instability steadily increases in both the cerebellum and cerebrum of ageing rats, and this process is much faster in the cerebellum.

Hydrolysis optimum of TCA Feulgen reaction on lymphocytes, liver cells, and Ehrlich ascites tumour cells

For simultaneous cytophotometric measurements of DNA and protein the Feulgen hydrolysis must be performed with trichloracetic acid (TCA) at room temperature because of the preservation of nuclear proteins. Already after 10 h of hydrolysis, a good discrimination between the frequency peaks in the histogram is obtained. The best results are achieved after a hydrolysis time in the range of the maximum of the hydrolysis curve (20 h). At this time, little variations of hydrolysis duration scarcely influence the cytophotometric data.

Cytofluorometric nuclear DNA determinations on human corneal endothelial cells

In order to investigate polyploidization of human corneal endothelium, nuclear DNA cytofluorometry was performed. Touch smears of 31 human corneal endothelium taken from 21 autopsies and 10 donor eyes were fixed with absolute methanol and subsequently stained by the pararosaniline-Feulgen nuclear reaction. The optimum condition for Feulgen hydrolysis of the specimens was determined, and all smears were hydrolysed in 1 n HCl for 5 min at 60°C. After the Feulgen reaction, the pararosaniline-DNA fluorescence was measured with an epi-illumination cytofluorometer (Olympus AH-QRFL). Polyploid nuclei, which are the result of abortive cell division after DNA synthesis, appeared with DNA contents of tetraploid (19 cases) and octaploid (two cases) classes. All other nuclei had DNA quantities within the range of the diploid class. Nuclei containing DNA between diploid and tetraploid amounts were not found. It was concluded that most human corneal endothelial cells are of post mitotic G1 population but that a few are in the generative G0 phase.

Cytochemical analysis of the chromatin of liver cell nuclei and nuclei of oval cells by means of the Feulgen hydrolysis curve in rats treated by thioacetamide.

The present study is an analysis of the Feulgen hydrolysis characteristics in nuclei of liver cells and oval cells in rats treated by thioacetamide (TAA) and of liver cells in control rats. The curves show a double-peaked pattern. A slower hydrolysis is noted in the first peak after TAA treatment. This suggests an alteration of the acid-labile part of the chromatin. The curve obtained in the oval cells is different from the one in the liver cells. The implications of these differences are discussed with respect to development of cholangiocarcinomas.

Cytochemical properties of euchromatin and heterochromatin

Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.

A semi-automated procedure for aiding the diagnosis of cervical neoplasms based on the measurement of acid-labile DNA in exfoliated cells.

Measurements of acid-labile fractions of DNA in cells exfoliated from cervical neoplasms (invasive squamous-cell carcinomas) using Feulgen techniques have shown elevated values in both abnormal and cytologically normal cell nuclei. These increased values occur early in the hydrolysis profiles before the Feulgen hydrolysis optimum for the cell nuclei of normal patients has been attained. They are of sufficient magnitude to enable a microcomputer programmed in BASIC to distinguish between normal and abnormal cells, even when the latter are of cytologically normal appearance. By semi-automating the measurement procedure using a microcomputer interfaced with a Vickers M86 microdensitometer, the assessment of acid-labile DNA in cell nuclei is proposed as a useful aid to the existing cytological diagnosis of cervical neoplasms.

N-banding in Triticum aestivum following feulgen hydrolysis

Terminal and/or interstitial N-bands were produced on the seven B-genome chromosomes and chromosomes 4 and 7 of the A-genome of Triticum aestivum cv. 'Chinese Spring' by a modified BSG technique following a standard Feulgen preparation. The banding was accomplished by modifying the barium hydroxide treatment.