Immunocytochemical localization of basic fibroblast growth factor in squamous cell carcinomas of the head and neck.

Angiogenesis is crucial for tumour growth and formation of metastasis. Basic fibroblast growth factor (bFGF) is known to have potent angiogenic activity and has been identified in a wide variety of malignancies including head and neck squamous cell carcinomas (HNSCC). Clarification of its localization in HNSCC is important for the understanding of angiogenesis. Cryosiat sections of 27 HNSCC were immunostained for bFGF using a standard streptavidin-biotin complex procedure. Western blot analysis revealed three immunoreactive bFGF isoforms of 18, 22 and 24 kDa. Immunohistochemical and -cytochemical localization of bFGF was studied at light and electron microscopic levels. bFGF was mainly localized within the focal tumoral areas rather than in the tumour stroma. On a subcellular level, the ultrastructural investigation showed electron-dense bFGF localization in the cytosol of the carcinoma cells, but bFGF labelling within the nuclei of HNSCC cancer cells was one prominent finding of this study. In conclusion, the presence of bFGF isoforms in most of the cancer cells supports the theory of a direct paracrine mechanism of bFGF from cancer cells on tumour angiogenesis. The nuclear localization of bFGF in the HNSCC cells supports its activity as a transcriptional factor.

Toxicity of 4-chloroaniline in early life stages of zebrafish (Danio rerio): II. Cytopathology and regeneration of liver and gills after prolonged exposure to waterborne 4-chloroaniline.

Ultrastructural alterations in liver and gills of embryonic and larval zebrafish (Danio rerio) following prolonged exposure to waterborne 0.05, 0.5, and 5 mg/L 4-chloroaniline for up to 31 days as well as after a 14-day regeneration period were investigated by means of light and electron microscopy. Acute toxicity was also tested at 25 and 50 mg/L. Survival of zebrafish embryos and larvae was only impaired from 25 mg/L 4-chloroaniline, but-after a transient stimulation following exposure to 0.5 mg/L-4-chloroaniline hatching was retarded after exposure to >/=5 mg/L, and fish displayed increasing rates of abnormal development and pigmentation. In contrast, hepatocytes displayed a time- and dose-dependent response from 0.05 mg/L 4-chloroaniline, including changes in nuclei, mitochondria, peroxisomes, endoplasmic reticulum, Golgi fields, lysosomes, and hepatic glycogen and lipid stores, as well as invasion of macrophages. In gills, dose-dependent effects were evident from 0.5 mg/L 4-chloroaniline and included deformation of secondary lamellae due to vacuolization and desquamation of respiratory epithelial cells in conjunction with dilation of intercellular spaces. Respiratory epithelial cells displayed progressive mitochondrial changes, induction of cytoplasmic myelinated structures, augmentation of lysosomes, and modifications of Golgi fields. Erythrocytes were severely deformed. A 14-day regeneration period was sufficient for almost complete recovery of pathological symptoms in both liver and gills. Only minor volumetric changes in hepatocellular organelles and a limited number of myelinated bodies, lysosomes, and cytoplasmic vacuoles were reminiscent of prior 4-chloroaniline exposure. In both qualitative and quantitative terms, most effects in hepatocytes after exposure of embryonic and larval zebrafish to waterborne 4-chloroaniline are comparable to the reaction of hepatocytes in adult zebrafish liver after prolonged sublethal exposure as well as in larval zebrafish after microinjection. Morphological changes in erythrocytes indicate disturbance of respiration as an additional mode of action of 4-chloroaniline.

Toxicity of 4-chloroaniline in early life-stages of zebrafish (Brachydanio rerio): I. cytopathology of liver and kidney after microinjection.

In addition to survival and hatching parameters, cytological alterations in liver and kidney of 4- and 6-d old zebrafish larvae (Brachydanio rerio) following single microinjection of fertilized eggs at the germ-ring stage with 5, 12.5, and 25 ng 4-chloroaniline/egg were investigated by means of electron microscopy. Whereas survival remained unaffected, microinjection with 4-chloroaniline disturbed hatching of larvae. Hatching was delayed by microinjection of 12.5 ng 4-chloroaniline/egg and above when compared to controls. Cytological investigations revealed ultrastructural changes in both liver and kidney in a dose- and time-dependent fashion. In the liver, major cytopathological changes included fenestration, fragmentation, and vesiculation of the rough endoplasmic reticulum, proliferation of atypical mitochondria, and atypical lysosomes. Furthermore, myelin whorls, lipid inclusions, and cholesterol crystals were increased, whereas glycogen stores were reduced. Renal tubular cells displayed altered brush borders, proliferation of nucleoli, atypical mitochondria, fenestrated, fragmented, and vesiculated RER cisternae, as well as giant lysosomes. Most of these effects indicate cellular dysfunction (e.g., disturbance of lipid metabolism in the liver), whereas others illustrate general cellular stress-responses to chemical aggression. Comparisons of results with those of previous studies based on conventional fish exposure prove the suitability and sensitivity of microinjection bioassays with zebrafish eggs as an alternative to conventional early life-stage tests.

Segment specificity of the cytological response in rainbow trout (Oncorhynchus mykiss) renal tubules following prolonged exposure to sublethal concentrations of atrazine.

Cytopathological alterations in proximal (PS I, PS II) and distal segments (DS) of rainbow trout (Oncorhynchus mykiss) renal tubules following exposure to 0, 10, 20, 40, 80, and 160 micrograms/liter atrazine for 4 weeks were investigated by means of electron microscopy. Cellular responses were clearly dose-dependent with a gradual increase in variability and intensity of effects. Ultrastructural modifications in PS I and II were observed from 10 micrograms/liter atrazine, in DS from 20 micrograms/liter. In PS I, major changes included proliferation of smooth endoplasmic reticulum, atypical mitochondria and lysosomes, as well as gradual alterations of the apical plasmalemma. Typical changes in PS II cells were a proliferation of peroxisomes and ring- and cup-shaped mitochondria, as well as alterations in the basal labyrinth; DS cells were characterized by a proliferation of atypical mitochondria with longitudinally oriented cristae, disorganization of Golgi fields and vacuolization of the cell base. Results document that different segments of the renal tubule in rainbow trout react not only with different levels of sensitivity to atrazine exposure, but also in a segment-specific way. Moreover, comparison of effects induced by atrazine with those resulting from chronic exposure to the herbicide linuron revealed a distinct substance specificity in the reaction of different tubular segments.