G. Nöhammer

Quantitative cytospectrophotometric studies on protein thiols and reactive protein disulphides in samples of normal human uterine cervix and on samples obtained from patients with dysplasia or carcinoma-in-situ.

Quantitative microspectophotometric studies have been made on sections of human cervix after staining for reactive protein thiol-groups (PSHr), and the sum of protein thiols with so-called reactive protein disulphides (together abbreviated as TRPS). Measurements were made on normal epithelium, apparently normal epithelium adjacent to a pathological lesion, dysplastic epithelium, carcinoma-in-situ, and adjoining stroma. The numbers of cases studied were: normal healthy controls (53); patients with dysplasias (34) and patients with carcinoma-in-situ (29). In the normal control sections the ratio of PSHr in epithelium:stroma was approximately 2.7 and this ratio was strongly decreased in dysplasias (1.6) and carcinoma-in-situ (1.5); the 3 populations of values had sufficient overlap to prevent this measurement being an effective discriminator. No significant variations were observed with TRPS-values except with changes in the stroma adjacent to apparently normal epithelium. However, the ratio of PSHr:TRPS was effectively discriminatory when this double-staining ratio was calculated for epithelial values:stromal values. These results are discussed in relation to the importance of thiol-groups in cell division and cancer, and the biological implications of similar changes observed in neighbouring apparently normal epithelium.

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 μm2) 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.

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

SummaryPrevious 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.

Histophotometric Quantification of the Field Effect and the Extended Field Effect of Tumors

Histophotometric investigations have been made on samples of human skin. Fresh frozen serial sections were fixed and stained for either reactive protein thiols (PSHr) or total reactive protein sulphur (TRPS) using modifications of the DDD-Fast blue B-method. In addition, total protein thiols (PSHt) were stained with the Mercurochromcyanide-method, and proteins were stained using a modified amido-black procedure. Significant differences were found between the different tumours investigated and normal tissue, and also between apparently normal tissue adjacent to the tumours and normal tissue from patients without tumour. To reveal such tumour-related changes of apparently normal tissue, termed the field effect of tumours, a double quotient had to be calculated from the PSHr-and TRPS-values determined from both epithelium (epidermis) and connective tissue. In addition, abdominal skin was investigated from patients without tumour and patients with tumours of the female genital tract, liver or breast. With the aid of the double quotient procedure, highly significant differences were found between normal abdominal skin of patients without tumours versus similar samples taken from patients with tumours. The tumour-related changes found with abdominal skin distant from the tumours have been termed the extended field effect of tumours. These general tumour-related changes, independent of the size, state or degree of malignancy of the distant tumour, could be shown to be due to changes in abdominal dermis.

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

SummaryConditions 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/105 g protein compared to 8.5 moles AB/105 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 μm 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 (Nöhammer 1978; Nöhammer 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.

Histophotometrical investigations on the contents of protein and protein thiols of the epithelium and stroma of the human cervix

SummarySections of 10 μm thickness of normal epithelium of the cervix uteri were stained with DDD-Fast Blue B for reactive protein thiols (PSHr) and with Amidoblack for proteins. Areas of approximately 0.3 mm2, both of the intact epithelium and of the adjacent stroma were scanned at 560 and 620 nm respectively, using a measuring spot of 10 μm ∅ and the average densities per unit area (E/μm2), of PSHr and proteins, were determined. No essential differences were found between the four different cell layers (basal (B), parabasal (P), intermediate (I) and superficial (S)) either with respect to PSHr or to the protein area densities. Thus it seems to be justified to introduce E/μm2-values for the epithelium as a total: 0.33 for PSHr and 0.40 for protein and to compare them with the corresponding values of the stroma: 0.13 and 0.23 respectively. The epithelium contains approximately 2.5 times more PSHr but only 1.7 times more proteins than the adjacent stroma, indicating that the greater PSHr-content of the epithelium is caused not only by the greater content of epithelial proteins but also by the greater PSHr-content of the epithelial proteins. The approximately equal densities per unit area (E/μm2) of the PSHr in the four epithelial cell layers, however, do not give any information about the intracellular localisation of PSHr. The investigation of individual single cells in the sections with a resolution of a 1 μm-∅ measuring spot shows that in the actively proliferating B- and P-cells nuclear PSHr make up 60 and 80% respectively of the total PSHr present, but only 5% in the non-dividing S-cells where considerable percentages of PSHr were found not only in the cytoplasm (46%) but also in the outer cell membrane (28%) and in the intercellular substance (22%). In summary, it can be said that the higher content of PSHr in the epithelium compared with the adjacent stoma points to a greater extent of the SH-controlled processes in the epithelium; these might be processes in connection with cell proliferation in the stratum germinativum, whereas in the superficial cell layer, functions of the outer cell membrane and of the intercellular substance might be relatively more important.

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

SummaryFixed 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 (ε367=21 000) were 1.893±0.072 (P<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.

Histophotometric quantification of protein and reactive proteinthiols in invasive carcinomas of the skin.

SummaryFrozen sections cut from 14 samples of invasive carcinomas of the skin were stained with Amido black for protein determination and with dihydroxydinaphthyldisulphide fast blue to quantify reactive protein thiols (PSHr) and were then analysed microphotometrically. It was found that all of the samples exhibited significant reductions in protein levels (49%–74%) and PSHr levels (32%–53%) as compared to normal epidermis. Thus, the content of proteins of PSHr groups was 1.7 times greater in the malignant tissue examined than in normal epidermis.These results are in accordance with those previously obtained in basal-cell epitheliomas.