Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane matrix promotes three-dimensional reorganization.

Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.

Modification of the protein A-gold immunocytochemical technique for the enhancement of its efficiency.

A modification in the protein A-gold immunocytochemical technique has been introduced for amplification of the labeling. This modification consists of performing additional incubation steps with an anti-protein A antibody and the protein A-gold complex. The original antigen-antibody-protein A-gold complex was further incubated with an antibody directed against protein A and then, in a fourth step, again with protein A-gold. This multiple-step protocol results in significant enhancement of the original signal. The modified technique can be applied to either light or electron microscopy protein A-gold immunocytochemistry. The advantage of such an approach is double: it allows for either amplification of the labeling when the original signal is of low intensity or use of highly diluted antibody solutions. The modification introduced was thus found to significantly enhance the efficiency of the technique.

Dose-dependent genotoxic effects of styrene on human blood lymphocytes and the relationship to its oxidative and metabolic effects.

Although the genotoxic potential of styrene is known, very limited information is available regarding its dose-dependent genotoxic response to human blood lymphocytes and how such response correlates with different metabolic events in whole blood lymphocytes. The present study was therefore carried out to study such a relationship using in vitro human blood lymphocytes from healthy volunteers. To study genotoxic response to styrene, sister chromatid exchanges (SCEs), cell cycle, and cell survival were analyzed. Lymphocytes were cultured for 72 hr in the presence of different concentrations of styrene (0-1,000 microM). Twenty-four hr before harvest, BrdU (5 micrograms/ml) was added to assess the increase in SCEs and cell cycle delay. Both the SCE frequency and the cell cycle length were increased linearly with increasing concentrations of styrene up to 200 microM, without addition of any exogenous metabolizing system. Above 200 microM, no further increase in genotoxic response occurred. The range of concentrations (10-200 microM) at which increase of cell cycle length due to styrene was observed did not impair the viability of the cells, suggesting that such cell cycle delay is a genotoxic-related event and not caused by cytotoxicity. In vitro metabolic transformation of styrene in whole-blood lymphocyte cultures without the presence of any exogenous metabolic activation system showed the formation of a reactive intermediate, styrene 7,8-oxide, to be capacity-limited, as verified from a nonlinear increase in the formation of styrene glycol. The value of such metabolic parameter reached a plateau above 200 microM styrene. The same phenomenon of saturation has also been observed with regard to other metabolic effects due to styrene in whole blood lymphocytes in culture, such as dose-dependent increase in lipid peroxidation and depletion of blood lymphocyte glutathione. Based on the relationship between the formation of different metabolic events and the genotoxicity of styrene, it may be possible that the genotoxic properties of styrene in human blood lymphocytes may be mediated initially not only by the formation of the presumably reactive styrene 7,8-oxide, but also by that of a reactive oxygen species as well. However, the present data are not sufficient enough to definitely identify the role of reactive oxygen species in such toxicity and therefore it warrants further study.

Discrimination between precancerous and cancerous lesions of the uterine cervix by DNA measurements on tissue sections.

Morphologically typical uterine cervical biopsies were separated into normal cervices, condylomas and cervical intraepithelial neoplasias (CIN) grades I, II and III. At least 100 nuclei per lesion were measured on 4 micron Feulgen-stained sections using a Zeiss microspectrophotometer, with a variant of the plug method used to compute the nuclear DNA content. DNA distribution histograms were then decomposed into subsets of diploid, tetraploid, octoploid and aneuploid cells. The decomposition, which assumed a log-normal model of polydiploidy distribution, led to the identification of six indices: (1) the percentage of diploid cells, (2) the percentage of tetraploid cells, (3) the percentage of octoploid cells, (4) the percentage of aneuploid cells with DNA contents less than tetraploidy, (5) the percentage of aneuploid cells with DNA contents between tetraploidy and octaploidy and (6) the percentage of aneuploid cells with DNA contents greater than octoploidy. These indices, along with the mean nuclear radius, the 5c exceeding rate and the 2c deviation index, generated a nine-dimensional space. Two methods of discriminant analysis on this space showed discriminating powers of 78.22% and 87.13%, respectively, as compared to the original diagnoses. The most discriminating variable in both analyses appeared to be the percentage of octoploid cells.

A review of the study of protein secretion applying the protein A-gold immunocytochemical approach.

Exocrine and endocrine types of secretion were investigated in various cells by applying the protein A-gold immunocytochemical approach. Several proteins secreted by rat pancreatic and parotid acinar cells, mouse ameloblasts, rat pancreatic B cells and lymph-node plasma cells, and frog hepatocytes were studied using specific antibodies. While light microscope immunohistochemistry has allowed for good topographical identification of positive cells in tissues, the protein A-gold approach used at the electron microscope level has demonstrated the presence of specific antigenic sites in particular cellular compartments. All secretory proteins studied were detected in the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules of the corresponding secreting cells. In addition, some of the proteins were also found in lysosome-like structures. When good ultrastructural preservation of the cellular organelles was achieved, the labeling was revealed with very high resolution and precise localization. In such cases, we found labeling over transitional elements of the endoplasmic reticulum and in smooth vesicles in the Golgi area. The Golgi apparatus was subdivided into three compartments according to differences in labeling: the cisternae on the cisside, those of the trans-side and the trans-most rigid one. Quantitative evaluations of the intensities of labeling have allowed for 1) demonstration of the high specificity of the different labelings; 2) revelation of the existence of a gradient of increasing intensity that follows precisely the progress of the proteins along their secretory pathway; and 3) identification of intracellular sites where increments of protein antigenicity occur. Furthermore, they have revealed the existence of alterations in protein processing that occurred under experimental and pathological conditions. Double-labeling approaches were performed to demonstrate two different antigenic sites on the same tissue section by applying protein A-gold complexes formed by gold particles of different sizes. Protein A-gold immunocytochemistry has also been combined with cytochemical and radioautographic techniques. This review thus demonstrates that high-resolution quantitative immunocytochemistry can contribute significantly to the investigation of the intracellular processing of secretory proteins. It also illustrates the potential and versatility of the protein A-gold technique, which in combination with other procedures constitutes a powerful method in cell biology.

Cytogenetic effects of low-level exposure to toluene, xylene, and their mixture on human blood lymphocytes.

The present study was undertaken to investigate, both in vitro and in vivo, the genotoxic potential of short-term low-level exposure to toluene, xylene, and their mixture, for which information is limited at the present time. Five adult healthy white men were exposed for 7 consecutive hours per day over 3 consecutive days to 50 ppm toluene and 40 ppm xylene either alone or in combination in a controlled exposure chamber. Such an exposure was repeated three times at intervals of 2 weeks. Blood samples were taken before and after the termination of such exposure. Three different cytogenetic end-points were evaluated using peripheral blood lymphocytes: number of sister chromatid exchanges (SCEs), cell cycle delay, and cell mortality. No significant effects on SCEs, cell cycle delay, and cell mortality were observed following such exposure to toluene or xylene or their mixture. Similarly, exposure of human blood lymphocytes in vitro to either toluene (0-2.5 mM) or xylene (0-2 mM) or their mixture for 72 h did not result in any significant cytogenetic effects at lower concentrations, while at higher concentrations, only cell mortality was found to be significantly affected. Thus our present study indicates that simultaneous exposure to low levels (within the admissible limits) of toluene, xylene, or their mixture for a short period does not pose any potential mutagenic threat to humans.