Effects of deoxyribonucleosides and cell-stimulation on frequencies of ultraviolet-B-induced micronuclei in human peripheral blood lymphocytes.

In the present study the involvement of deoxyribonucleotides (dNTPs) in the clastogenicity of ultraviolet-B (UVB) in unstimulated peripheral blood lymphocytes (G(0)-PBLs) was investigated. This was studied by analyzing the frequency of UVB-induced micronuclei (MN), either after adding a cocktail of the four deoxyribonucleosides to the PBLs immediately after exposure to UVB, or by stimulating the cells before exposure. In total, PBLs obtained from two different donors were investigated. For both donors, it could be demonstrated that addition of deoxyribonucleosides to UVB-irradiated G(0)-PBLs resulted in a significant reduction of the clastogenic effect of UVB. A gradual reduction of the clastogenic effect of UVB could also be realized by irradiating PBLs that were progressively more stimulated with the lectin PHA before exposure. The latter finding is explained by upregulation of intracellular pool sizes of dNTPs in stimulated PBLs.

The induction and analysis of micronuclei and cell killing by ultraviolet-B radiation in human peripheral blood lymphocytes.

The DNA-damaging potential of ultraviolet-B (UVB) radiation was investigated by analyzing the frequency and origin of micronuclei (MN) in cytokinesis-blocked, binucleated (BN) peripheral blood lymphocytes (PBL) and cloning efficiencies (CE) of PBL after exposure to different fluences of UVB. In total, PBL obtained from five normal donors were investigated. The PBL from all donors showed a dose-related, linear-quadratic increase in the frequency of MN per 1000 BN cells and in the frequency of micronucleated BN cells. In two experiments the origin of UVB-induced MN was studied by analyzing MN for the presence or absence of centromeres by applying the MN assay in combination with a centromeric probe and fluorescence in situ hybridization. This revealed, for the first time, that UVB-induced MN were centromere negative, indicating that UVB acted exclusively as a clastogenic agent in the tested dose range. The PBL from all donors showed a clear dose-dependent decrease in CE, after UVB exposure. The UVB-exposed PBL from all donors showed an inverse relationship between the induction of MN and the decrease in CE, but regression analysis revealed no correlation between the induction of MN and the decrease in cell survival. It is concluded that UVB has a clastogenic and cytotoxic effect on PBL.

Involvement of treponemal surface-located protein and carbohydrate moieties in the attachment of Treponema denticola ATCC 33520 to cultured rat palatal epithelial cells.

We studied the nature of attachment of Treponema denticola ATCC 33520 to a microscopically distinct population of rounded rat palatal epithelial cells. The motility of the freshly harvested spirochetes appeared not be a prerequisite for attachment. Treatment of T. denticola ATCC 3350 with proteinase-K, heat, glutaraldehyde, formaldehyde and periodate oxidation decreased the attachment to the rounded rat palatal epithelial cells, indicating the involvement of protein and carbohydrate moieties. Trypsin treatment had no effect on the attachment. The attachment of T. denticola ATCC 33520 was decreased after treatment with native non-immune rabbit serum, native polyclonal rabbit serum, D-mannose, N-acetyl-D-galactosamine and sialic acid. The results indicate that the attachment of T. denticola ATCC 33520 to rounded rat palatal epithelial cells is mediated by trypsin-resistant adhesin(s) of protein and carbohydrate nature, with affinity for D-mannose, N-acetyl-D-galactosamine and sialic acid.

Attachment of T. denticola strains ATCC 33520, ATCC 35405, B11 and Ny541 to a morphologically distinct population of rat palatal epithelial cells.

In the present study an assay for the attachment of T. denticola to epithelial cells is described. An indirect immunohistochemical staining method, using two native polyclonal antisera, revealed dark-brown coloured spirochetes attached to rat palatal epithelial cell (RPE) monolayers. In addition, two morphologically distinct populations of RPE cells could be distinguished in the monolayers when using phase contrast microscopy. One minor population consisted of isolated rounded RPE cells that were lying on top of a confluent monolayer of flattened RPE cells. The rounded RPE cells were more receptive for the attachment of T. denticola than the flattened cells. The rounded RPE cells were evenly distributed over the monolayer, but the attachment of spirochetes to the rounded cells was greater at the edge than in the centre of the monolayers. The percentage of rounded RPE cells with attached spirochetes depended on the incubation time (optimum 6 h), temperature (optimum 37 degrees C) and pH (optimum 7.0). It is speculated that the attachment of T. denticola is a physical/chemical process of yet unknown nature and that differences in the number of microvilli and/or the amount of available receptors, between the two morphologically distinct cell types, accounts for the differences in the numbers of attached spirochetes.

Attachment of Treponema denticola strains to monolayers of epithelial cells of different origin.

The attachment of 10 different Treponema denticola strains to monolayers of 4 types of epithelial cells derived from rat palatal epithelium, guinea pig ear, human buccal epithelium and human corneal epithelium was screened microscopically. Most T. denticola strains were able to attach to all four types of epithelial cells. The T. denticola strains seemed to attach better to epithelial cells derived from primary cultured material. The T. denticola strains showed different degrees of attachment. Scanning electron microscopy studies revealed that the attachment of T. denticola was not only tip-associated but occurred also at random points in close contact with microvilli of the epithelial cells. Attached spirochetes were non-uniformly distributed over the monolayers, indicating the presence of receptive subpopulations of epithelial cells in the monolayers.

Hemagglutination activity of Treponema denticola grown in serum-free medium in continuous culture.

Hemagglutination by different Treponema denticola strains was observed for erythrocytes of human, horse, bovine, and rabbit origin. The growth of T. denticola ATCC 33520 in serum-free medium in continuous culture enabled us to study the hemagglutinating activity of freshly harvested spirochetes of a defined physiological status. The hemagglutinating activity was cell bound and not related to motility or appendages, such as fimbriae. The activity was destroyed by proteolytic enzymes, heat, and alkylation, indicating that the agglutinin is of a proteinaceous nature. In addition, periodate oxidation of the spirochetes indicated the involvement of carbohydrate groups. Microscopic inspection of the hemagglutination mixtures at the titration endpoints revealed that only a part of the spirochete population was involved in the hemagglutination process. The hemagglutinating activity was found to be growth phase related. The activity was blocked by serum, while of all tested amino acids and carbohydrates, only sialic acid blocked the activity at low concentrations. In conclusion, we found a hemagglutinating activity in T. denticola which was cell bound and growth phase related. The agglutinin may be a glycoprotein, like lectin, that recognizes sialic acid as a receptor.

In vivo induction of aryl hydrocarbon hydroxylase in human scalp hair follicles by topical application of a commercial coal tar preparation.

Five low-dose applications of a commercial coal tar preparation on a small scalp skin region resulted in an induction of aryl hydrocarbon hydroxylase (AHH) activity in freshly isolated human hair follicles. Large but reproducible interindividual differences in AHH-inducibility could be detected. The method offers the opportunity to measure AHH-inducibility, which has been correlated to the risk of developing chemical-induced cancer, in vivo in normal epithelium, a cell-type highly relevant for chemical carcinogenesis. Smoking habits did not have any effect on AHH-activity in freshly isolated hair follicles. Therefore the method potentially permits the identification of persons with high and low genetically determined AHH-inducibility.