Quantitative measurements of the changes in protein thiols in cervical intraepithelial neoplasia and in carcinoma of the human uterine cervix provide evidence for the existence of a biochemical field effect.

Quantitative micromethods have been used for measuring reactive protein thiols (PSHr), total reactive protein sulfur (TRPS), total protein thiols (PSHt), and protein disulfides (PDS) in fixed frozen sections of human uterine cervix. PSHr and TRPS were stained using 2,2'-dihydroxy-6,6'-dinaphthyl disulfide; PSHt and PDS were stained using mercurochrome methods. Microspectrophotometric measurements were made on the stained sections using a microdensitometer with associated data processing; the results obtained for areas of epithelium and stroma were converted to absorbance values per micron 2. Samples of uterine cervix that were diagnosed as containing cervical intraepithelial neoplasia (CIN) I-III or carcinoma were examined and compared with samples of normal uterine cervix. Measurements were made not only on identified lesions but also on apparently normal tissue obtained from the same cervix. Epithelial/stroma ratios (E/S) were calculated for PSHr, TRPS, PSHt and PSHt + PDS; in addition, the double ratios of PSHr/TRPS and PSHt/PSHt + PDS were also calculated for E/S. The mean E/S values for PSHr and PSHt were significantly different for all types of lesion compared with control samples. The E/S ratios for apparently normal tissue obtained from cervices with CIN or carcinoma were also significantly different compared with corresponding control values, indicating a field effect. There was a considerable degree of overlap between individual values in the control groups versus those obtained with each type of lesion. The corresponding mean E/S values for TRPS and for PSHt + PDS in the samples containing lesions were not significantly different from control means except for the group containing CaCx. However, the mean values for the double ratios (PSHr/TRPS and PSHt/PSHt + PDS) were significantly different in the groups containing lesions compared with the controls. Moreover, apparently normal tissue obtained from cervices containing CIN or carcinoma had different mean values compared with the controls, confirming the existence of a field effect. The degree of overlap of individual values in the lesion groups compared with the control values was much less with double ratio values than previously noted for single ratio values. In consequence, the double ratio measurements clearly discriminated CIN I + II and CIN-III from controls. Our data show that CIN is associated with marked changes in tissue protein thiols and disulfides and that these differences extend to neighboring apparently normal tissue indicative of a field effect.

Protein thiols in normal and neoplastic human uterine cervix.

Protein-thiol groups that react with dihydroxydinaphthyl disulphide during a 7 h incubation (so-called reactive protein thiols, PSHr) have been quantitatively measured on sections of human uterine cervix by microcytospectrophotometry. Measurements were made on areas (1 micron 2) of epithelium and adjoining stroma in samples of normal cervix, and in samples obtained from patients with dysplasia, carcinoma-in-situ and invasive cancer. The ratio of PSHr in epithelium to stroma is substantially reduced in the pathological conditions compared with normal and in apparently normal adjacent areas. Such changes in PSHr are discussed in relation to the redox balance of the tissue, and free radical disturbances previously described.

[Possibilities and limits of the quantitative demonstration of protein thiols with the DDD-reagent in cells of cervical epithelium (author's transl)]. / Möglichkeiten und Grenzen der quantitativen Bestimmung von Protein-Thiolen mit dem DDD-Reagens in Cervix-Epithelien.

From six patients, all with invasive carcinoma of the cervix uteri, two groups fo smear preparations, each consisting of eight samples, were taken: The first, group A, before and the second, group B, after mechanical removal of the discharge which was present in abundant quantities. All samples were treated for 24 h with DDD to reach the fast-reaching SH-groups, coupled with Fast Blue B and measured cytophotometrically at 560 nm. Without any exception, the neoplastic cells of group A showed significantly lower extinction values both of the nucleus and of the total cell, if compared to group B. The mean extinction difference amounts to 55% and is highly significant and some evidence is given that the viability of the cells plays an important role. For this reason, the quantitative evaluation of the DDD Fast Blue B does not seem to be useful for cytologic routine investigations.

The quantification of the histochemical protein staining with 2-hydroxy-1-naphthaldehyde (HNA) demonstrating primary amino groups of proteins.

The usefulness of the HNA-pH4-1d staining, which histochemically demonstrates primary protein amino groups under the conditions used, for the microphotometric quantification of proteins was investigated. A correlation (r = 0.986) has been found between the mean protein contents of fresh frozen and fixed sections prepared from different tissues of rats and the corresponding mean integrated extinction values determined histophotometrically after HNA-pH4-1d staining. A histophotometric extinction of E = 0.284 corresponded to 10(-12) g protein. The mean integrated extinction values determined cytophotometrically of different single cells and nuclei stained using the tetrazonium coupling method for proteins correlated (r = 0.989) with corresponding extinction values measured after HNA-pH4-1d staining. A cytophotometric extinction after HNA-pH4-1d staining of E = 0.130 correspond to 10(-12) g protein.

Histochemical demonstration of primary amino groups with 2-hydroxy-1-naphthaldehyde (HNA): optimization of the method.

2-hydroxy-1-naphthaldehyde (HNA) was used for the histochemical demonstration of primary amino groups. The Schiff-bases formed exhibited a double-banded maximum absorption at lambda = 420 nm and lambda = 400 nm, respectively. The dependence of the equilibrium of the Schiff-base formation upon the concentration of HNA, the reaction medium, especially upon its pH-value, was investigated with Ehrlich ascites tumour cells and rat liver parenchymal cells. Optimum conditions were elaborated for the histochemical reaction running either in absolute ethanol (HNA-EtOH) or in a mixture of ethanol and acetate buffer pH = 4 (HNA-pH4), the equilibrium of the reactions adjusted after 10 d and 1 d, respectively. The HNA-pH4-method can be used for the histochemical demonstration of primary alpha-, delta-, and epsilon-amino groups of proteins. The HNA-EtOH-method comprises primary amino groups of proteins and of other substances insoluble in absolute ethanol. Both histochemical methods proved to be reproducible.

[Quantitative cytospectrometrical determination of protein sulfhydryl groups and reactive disulfide groups by the DDD-Fast blue B-staining method on Ehrlich ascites tumor cells (author's transl)]. / Quantitative cytospektrometrische Bestimmung von Proteinsulfhydrylgruppen und von reaktiven Disulfidgruppen mit der DDD-Echtblau B-Färbund an Ehrlich-Ascites-Tumorzellen.

Protein sulfhydryl groups are histochemically demonstrated by reacting with DDD followed by coupling with Fast blue B. The molar absorptivity of the formed azo dye is 19000 per mole SH reacted. DDD simultaneously reacts with protein-SH- and -SS-groups. However, the reaction with SH-groups is approximately 1000 times faster than that observed with SS-groups. With Ehrlich ascites tumour cells the reaction of DDD with SH-groups is completed within 7 h while the reaction of DDD with SS-groups needs 14 days for completion. Due to the extreme difference in the reaction rates protein bound SH-groups as well as reactive SS-groups can be estimated quantitatively by cytospectrophotometrical methods. The cells investigated showed an average SH-content of (1,30 +/- 0,03) X 10(-14) M SH/cell while the average content of reactive SS-groups was (1,59 +/- 0,28) X 10(-14) M SS/cell. In addition it was found that especially the amount of reactive SS-groups per cell is not constant but exhibits seasonal variations.

[The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. I. Optimization of the staining method (author's transl)]. / Die cytospektrometrische Proteinbestimmung mit der Tetrazonium-Kupplungsreaktion. I. Optimierung der Färbemethode.

The tetrazonium coupling reaction using Fast blue salt B and the sodium salt of 6-hydroxy-naphthalinsulfonic acid-(2) serves as a method for histochemical demonstration of proteins. Fast blue salt B reacts with histidin, tyrosin and in a slighter extent also with tryptophan. It is shown in this paper, that the histochemical tetrazonium reaction carried out under the optimal conditions is a specific staining for proteins and highly reproducible. The maximum of absorption at lambda = 530 nm of the azo dyes generated by the coupling reaction followes the Lambert-Beer's law. Nucleic acids as well as protein-SH-groups do not react with Fast blue salt B under the conditions described. Due to the exact reproducibility and specifity the tetrazonium coupling reactions seems to be a suitable method for at least comparative, microspectrometric protein determination within one species of cells.

The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. II. Calibration of the staining method.

The protein content of identical samples from 5 different cell types (lymphocytes of the thymus and of the bursa of FABRICIUS of the chicken, EHRLICH ascites tumor cells, YOSHIDA ascites tumor cells, and rat liver cells) have been determined both macroscopically, by the method of LOWRY (1951), and microspectrometrically, by the tetrazonium coupling method of NOHAMMER (1978). In all the different cell types, a strong correlation is shown between the protein values determined by the 2 methods. By comparing the values thus measured, a conversion factor has been obtained such that an extinction of 0.4235, determined microspectrometrically, corresponds to a protein mass of 1 pgm. To test the accuracy of the microspectrometric method of protein determination at the lowest extreme point, the protein content of rat liver mitochondria has been measured and a mean value of 0.239 pgm protein per mitochondrion obtained. This corresponds well with the value of 0.233 pgm per mitochondrion as determined macroscopically by GEAR (1972).

Quantification of protein thiols in morphologically intact cells of the cervical epithelium. II. Effects of cell differentiation.

Protein-SH-groups (PSH) were determined quantitatively in basal (B), parabasal (P), intermediary (I) and superficial (S) cells of normal portio epithelium in order to obtain information about the correlation between PSH and cell differentiation. It was found that total PSH increase from B to S by 67 per cent, the main step occuring from B to P. Nuclear PSH drop continuously from B to S by 80 per cent. Thus, cytoplasmic PSH must raise (by 400%). The area of the increases strongly from B to S (by more than 1000%). Thus, differentiation is accompained by a drastic "dilution" of total PSH and PSH per area unit (1 micron2) both of nuclear and cytoplasm drop considerably. The findings about nuclear PSH are interpreted in terms of the loss of proliferative activity during differentiation, about cytoplamic PSH as expression of decreasing activities of PSH depending metabolic processes.

Microphotometrical quantification of protein thiols in morphologically intact cells of the cervical epithelium. I. Basic results with superficial cells of healthy women.

The investigations are based on Esterbauer's results according to which the staining of protein thiols (PSH) with DDD-Fast Blue B (Barrnett and Seligman) is of a quantitative order. Consequently, the PSH of the nucleus and the cytoplasm of superficial cells of the ectocervical epithelium of healthy women are determined to 0.3 and 4.1 X 10(-14) moles, respectively. After it has been found that the mean absorption spectrum between 310 and 238 nm of the pure proteins, the extinction at 278 nm of a cytoplasmic area (300 to 400 micron2) of unstained cells is taken as a first measure for the cytoplasmic protein content which is calculated to approximately 5 X 10(-18) moles per micron2 whereas the PSH content results to 1.9 X 10(-17) moles per micron2. From this value results a very plausible number of 3.6 SH per 10(5) g of cytoplasmic proteins.

Differential glucocorticoid effects on proliferation and invasion of human trophoblast cell lines.

Several clinical situations require continuous glucocorticoid (GC) treatment during pregnancy. A well-known deleterious side effect of such treatment is the higher incidence of growth-restricted fetuses, for which a too shallow trophoblast invasion is presently hypothesised as the underlying cause. This study investigated whether the synthetic GC triamcinolone acetonide (TA) influences proliferation, invasion and endocrine activity of human trophoblast. BeWo and JEG-3 choriocarcinoma cell lines both express GC receptors (western blotting) and were used as models for human trophoblast. JAR devoid cells of GC receptor were used as negative control. The cells were cultured for 48 h without (control) or with 0.5, 5 and 50 microM TA. In the presence and absence of serum, proliferation was determined by cell counting and measuring the cell cycle regulating protein cyclin B1 (Western blotting); invasion was determined by a conventional Matrigel invasion assay and by measuring the secretion (ELISA) of matrix-metalloproteinases (MMP-2, MMP-9) into the culture medium; endocrine activity was assessed by measuring the levels of human chorionic gonadotropin (ELISA) into the culture medium. TA altered the number of viable and dead cells as well as cyclin B1 levels and, to a lesser extent, invasion of BeWo and JEG-3, with a strong influence of serum. BeWo and JEG-3 cells reacted differently and in most instances reverse. In the cell lines used as models of human trophoblast, TA alter some functions relevant to proliferation and invasion, and suggest that caution should be exercised when treating women with GCs during pregnancy.

A modification of the amidoblack-TCA-staining for quantitative microspectrometrical determination of proteins in tissue sections.

Conditions are described by which cells and fresh frozen tissues, following fixation with ethanol-ether, and after staining with the amidoblack (AB)-TCA-staining method, show a modified dye/protein ratio of 0.9 moles AB/10(5) g protein compared to 8.5 moles AB/10(5) g protein ( Schauenstein et al. 1980) as in a previously used method. In contrast to the AB-TCA-method, which leads to extremly high and unmeasurable extinctions in tissue sections, staining with the modified AB-TCA- 23st -method with 10 microns tissue sections produces easily measurable extinction values. A correlation of the microspectrometrically determined mean total extinction values of different cell types and nuclei after staining with the tetrazonium method (N ohammer 1978; N ohammer and Desoye 1981) and on the other hand with the AB-TCA 23st -method has been found. The microspectrometrically determined extinctions after AB-TCA 23st -staining can be calculated; an extinction of 0.04248 corresponds to 1 pgm protein.

Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B.

Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc = 0.999). The maximum absorbance measured after 2,4-DNPH-staining (epsilon 367 = 21,000) were 1.893 +/- 0.072 (P less than 0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.

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.

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). The variety of DDD-staining methods demonstrated on Ehrlich ascites tumor cells.

2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specificity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhammer (1978) and calibrated by Nöhammer et al. (1981), was used.

The microspectrometrical determination of the nucleus-total-cell-protein-ratio: a possibility of objective cell type analysis.

Cells from smears of the normal human squamous epithelium of the gingiva, fixed and stained for protein using the tetrazonium method optimized by Nöhammer and calibrated by Nöhammer et al., were investigated. The extinctions of both the total cells and of the rectangular areas circumscribing the nuclei, were measured microspectrometrically. Altogether 417 cells from 6 healthy persons of both sexes were investigated. 9 distinct subgroups of cells were found showing an exact linear correlation between nuclear and total cell extinctions. In the graph of both the nuclear and the total cell extinctions the 9 subgroups can be seen as 9 distinct linear groups of points, defined exactly by their regression lines. Thus, every squamous epithelial cell within the smear can be typed definitely and objectively in respect to its membership of one of these 9 linear groups of points. The obviously definite, legitimate connection between the extinctions of the total cells and of their nuclei affords a glimpse into the processes of cellular differentiation and allows the definition of the so-called stem cell in terms of protein content of the total cell and of the nucleus.

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides.

Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is epsilon 520 = 34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells.