Vital cell labelling for the detection of invasive growth in the chick embryo skin invasion assay.

Of the various in vitro invasion assays described, only a few use tissues as substrates, for example, the chick heart fragment assay and the chick embryo skin (CES) invasion assay (Noguchi et al., 1978). We have improved culture conditions for the CES invasion assay (Schlage, 1989). A suspension of neoplastically transformed cells is incubated on an explanted piece of skin from a 9-10-day-old chick embryo. After 1, 2 and 3 days, the explants are fixed and cross-sectioned. As a measure of invasiveness, the number of invading cells and their mitotic activity should be evaluated. We tested the suitability of the vital fluorescence dye PKH-2 (Horan and Slezak, 1989) for improving discrimination between CES cells and invading cells; human cervical carcinoma (HeLa), 3-methylcholanthrene (MCA)-transformed mouse embryo fibroblasts (10T1/2-MCA7), and MNNG-transformed rat tracheal epithelial cells (RTE-MNNG). The three cell lines formed distinct infiltrates on days 2 and 3, but on day 1, only 10T1/2-MCA7 and RTE-MNNG cells formed infiltrates. Although PKH-2 labelling was found suitable for the detection of invasion, cellular resolution and dye stability in cryosections is still unsatisfactory. To overcome this for routine work, we suggest improving the histological processing and using PKH-26, which is a more stable dye.

Two-Stage Transformation Assay for Cigarette Smoke Condensates using Murine c3h-10t1/2 Fibroblasts.

We investigated whether the two-stage transformation assay can be applied in routine testing for promoter-like activity of cigarette smoke condensate (CSC) as an in vitro equivalent of an in vivo tumorigenicity assay (mouse skin painting). We adopted a published assay procedure (Frazelle et al., 1983a), using 3-methylcholanthrene (MCA, 0.37mumol/litre, 24hr treatment) as the initiator. Rigorously standardized experimental conditions, such as multiparameter-screened serum, one fixed subculture level, and a rigid preculturing schedule, were employed. Transformation was expressed as the fraction of dishes containing type II and type III foci. Compared to the positive control, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), transformation responses to CSC (three CSC batches that had different in vivo activity) were lower. Variations in dose-response relationships did not allow distinction between two of the three CSC batches, even with data pooled from seven assay repetitions over 2 years. In a second approach, to enhance the assay resolution, that is, the signal-to-noise ratio, promoter treatment twice per week was ineffective: the response and the background were both increased. Lowering the initiator concentration (0.08mumol/litre) enhanced the signal-to-noise ratio for TPA, but not for CSC. Even after standardization and enhancement of sensitivity, the two-stage transformation assay is useful primarily for qualitative assessment of promoter-like activity of weak promoters, such as CSC, rather than for quantitative comparisons.

Use of the HET-CAM Test for the Determination of the Irritant Potential of Cigarette Sidestream Smoke.

We investigated the suitability of the HET-CAM test for the determination of the irritant potential of cigarette sidestream smoke (SS). Coded test solution (0.2ml) was applied to the chorioallantoic membrane (CAM) of 10 day-old White Leghorn chick embryos. The time from the start of exposure to the occurrence of blood vessel injection (I), haemorrhage (H) and coagulation (C) in the first 5 minutes was recorded, and irritancy scores (IS) from 0 to 21 were calculated (Luepke, 1985). For the microbicide Kathon CG((R)) as positive control, a dose-dependent increase in irritancy between 0.3 and 15g/litre was seen (IS up to 17). Mainstream smoke (MS) or fresh SS from the Reference Cigarette 2R1 was passed through phosphate buffered saline (PBS) in a glass impinger. MS-PBS concentrations between 1.7 and 10 puffs/ml elicited IS from 4.5 to 9. SS-PBS concentrations between 4.5 and 15 puffs/ml elicited IS from 3 to 7.5, an underestimation due to mortality associated with an immediate breakdown of blood circulation through the CAM vessels, thereby interfering with the irritation parameters I and H. We propose to test blood circulation-independent irritation endpoints, for example trypan blue uptake in the CAM (INVITTOX/ERGATT/FRAME, 1996), for determination of the irritancy of SS.

Tobacco smoke-induced alterations of cytokeratin expression in the rat nasal activity cavity following chronic inhalation of room-aged sidestream smoke.

In a 12-month inhalation study on rats using room-aged sidestream smoke (RASS, 12 microg total particulate matter (TPM)/l) as an experimental surrogate for environmental tobacco smoke (ETS), we investigated differentiation changes, i.e. altered cytokeratin (CK) expression, in the epithelial lining at nasal cavity level 1 (NL1) (anterior portion of nasal cavity), and their correlation with histomorphological changes. In addition to conventional histopathological examination, routine paraffin sections were immunohistologically stained for various rat CK and evaluated. Changes in CK expression were observed in the nonciliated respiratory epithelium of maxilloturbinate, lateral wall, and nasoturbinate: in basal cells, increase of CK14 and CK18 and decrease of CK15; in nonciliated columnar cells, increase of CK15 and CK19. These CK changes had histomorphological correlates, i.e. reserve cell hyperplasia and squamous metaplasia. CK expression changes were also seen at sites without histomorphological changes, e.g. enhanced expression of CK14, CK18 in ciliated cells at the dorsal meatus, and CK15 at the septum. Most of the CK expression changes seen after 1 year of RASS exposure resembled the changes previously seen after 8 days of exposure.

Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. II. Changes in cytokeratin expression patterns following 8-day exposure to room-aged cigarette sidestream smoke.

The expression of specific cytokeratin (CK) polypeptide patterns is a sensitive marker of the cytoskeletal differentiation of epithelial cells. We developed an immunohistochemical method to assess CK expression patterns in the rat respiratory tract using serial paraffin-embedded sections from the nasal cavity, trachea, and lung. In the present study, this method was used to detect exposure-related differences in CK expression patterns in adult Wistar rats following inhalation of room-aged sidestream smoke (11 mg total particulate matter/m3 air, 8 days, 12 hr/day, whole body). In the anterior nasal cavity level 1 (NL1), changes in CK expression patterns were observed in the respiratory epithelium of the lateral wall and the maxilloturbinate (CK14, CK15, and CK18) and in the squamous epithelium of the ventral meatus (CK13). At nasal cavity level 2 (NL2), immediately behind NL1, changes were observed in the olfactory epithelium (CK13, CK14, and CK18) and in the respiratory epithelium of the septum (CK7 and CK19), the lateral wall (CK7 and CK13), and the lateral aspect of the maxilloturbinate (CK14). Changes were also observed in the submucosal glands, nasolacrimal duct, and vomeronasal organ. In the trachea only CK7 expression changed, and in the lung expression of CK7 (bronchioli) and CK8 (bronchus) changed; the expression of other CK polypeptides did not change. The observed changes in CK expression at NL1 correlated with the histomorphological changes, whereas CK expression changes were also seen in the olfactory and respiratory epithelia at NL2 and in the trachea and lung, where no histomorphological changes were seen. These findings indicate that changes in CK expression in respiratory tract epithelial cells are a sensitive marker for cellular stress response.

Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. I. Determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung.

Cytokeratin (CK) polypeptides constitute the intermediate filament cytoskeleton of epithelial cells. The patterns of CK expression can be regarded as specific markers for the epithelial differentiation status. Our objective was to map the cell type-specific CK expression patterns at all representative sites of the respiratory tract of untreated rats to use as a base for the detection of inhalation exposure-related differentiation changes. Using routine paraffin-embedded sections and a panel of well-characterized monoclonal antibodies for immunohistochemistry, we obtained CK staining patterns as follows. Nasal cavity: respiratory epithelium CK18, CK19 (basal, ciliated, nonciliated cells), CK14, and/or CK15 (basal and nonciliated cells); olfactory epithelium CK18 (basal, mid, apical zones and Bowman's glands), CK14, and CK15 (basal zone); squamous epithelium of ventral meatus CK14, CK15 (basal and suprabasal cells), CK1, 10/11, and CK13 (suprabasal cells); glands and columnar epithelia of vomeronasal organ and nasolacrimal duct CK7 and CK13 in addition to respiratory epithelial CK pattern. Trachea: similar to nasal respiratory epithelium with pronounced CK15 and additional CK7. Larynx: CK14, CK15 (basal, ciliated, nonciliated cells), CK8, CK18, CK19 (not in basal cells), CK4, and CK13 (cuboidal and squamoid cells of ventral half). Lung: bronchial epithelium CK14 and CK15 (basal cells only); bronchial and alveolar epithelium CK7, CK8, CK18, and CK19; bronchiolar epithelium similar but less CK8 and no CK7; pleural mesothelium CK7, CK8, and CK19. This inventory of complex CK expression patterns provides the basis for investigating test substance-related effects in inhalation toxicology, e.g., cigarette smoke-induced changes.

Cytokeratin patterns of epithelial cells of the rat nasal cavity in vivo and in vitro.

Nasal epithelial cells are a primary target for the actions of inhaled substances. To enable the determination of alterations in cell differentiation in the rat inhalation model, we developed a methodology to assess cytokeratin expression in rat nasal tissue. A panel of commercially available antibodies was validated for specificity to defined rat cytokeratins by immunoblotting. The development of immunohistological procedures to enhance spatial resolution enabled mapping of cytokeratin patterns in various cell types at defined regions and levels of the rat nasal cavity using serial sections and a standardized evaluation schedule.

[Studies of the metabolism and excretion of silybin in the rat]. / Untersuchungen zur Verstoffwechselung und zur Ausscheidung von Silybin bei der Ratte

The metabolism and excretion of silybin (as N-methyl-glucamine salt) was investigated after intravenous and oral administration to rats. In the urine, silybin was excreted mostly in the unchanged form after intravenous as well as oral application, whilst in the bile it appeared above all in the form of metabolites. By hydrolysis with arylsulfatase/beta-glucuronidase, the metabolites were identified as sulfate and glucuronide conjugates of silybin and dehyrosilybin; the latter appeared in small quantities as a dehydrated product of silybin. After intravenous injection of 20 mg silybin per kg body weight, the excreted amount of silybin after 48 h was 8%, whereas 76% was eliminated in the bile within the same period of time. After oral application of 2--20 mg silybin/kg body weight 20% after 40 mg/kg 35% and after 120 mg/kg 20% of the administered silybin was excreted in the bile during 48 h. The maximum excretion rate was achieved at application of 20 mg/kg p.o. after 1 h. At this dosage, 2--5% was eliminated within the same time in the urine. The excretion of silybin mainly took place (more than 80% of the total of excreted bilybin) in the bile, both after oral and intravenous administration.