Ultrastructural changes in chemically induced preneoplastic focal lesions in the rat liver: a stereological study

Ultrastructural changes were investigated and quantified, using a stereological approach, in early gamma-glutamyl transpeptidase (GGT)-positive focal lesions, induced in the rat liver by treatment with a single initiating dose of diethylnitrosamine (DENA) followed by promotion with phenobarbitone (PB) for 30 weeks. Within the extra-hepato cyte environment of focal tissue, the mean volume occupied by Ito cells was markedly decreased, whilst that occupied by endothelial and Kupffer cells was increased, when compared to uninvolved tissue from the same rat livers. The bile canalicull were dilated, but nosignificant differences in the mean volume occupkd by the sinusoidal and Disse spaces were noted. In focal hepatocytes there was a striking overproduction of lipid droplets and proliferation of smooth endoplasmic reticulum (sER). Whorls of concentrically arranged, parallel ER membranes were found only in the hepatocytes of preneoplastic foci, in association with the proliferated sER, and never in the surrounding, uninvolved tissue. The Increase In mean volume of the sER, lipid droplet and cytoplasmic matrix compartments, together with the appearance of whorls, were the major contributing factors to the marked hypertrophy seen in focal hepatocytes. The mean volume of the rough endoplasmic reticulum, mitochondrial, lysosomal, peroxisomal and nuclear compartments per hepatocyte also Increased, but contributed to a lesser extent to the cellular hypertrophy. It is speculated that whorls may be structural adaptations, resulting from a possible alteration in the normalfeedback control of cholesterol synthesis, for the production of sterols and the biogenesis of sER in eosinophilic-type focal cells. The significance of changes observed in focal tissue, and the high biological variation noted between foci, is discussed in relation to the hepatocarcinogenic proces

Application of the New Stereological Probes to the Study of the Melanosome in Cloudman S91 Melanoma Cells

The relationship between melanosome size and number and melanin content has been investigated in Cloudman S91 melanoma cells growing in vitro using both “model-based” and “design-based” stereological procedures. Cells were cultured for 4 days, harvested at daily intervals, and resin-embedded for light and electron microscopy; one aliquot of each sample of cells was assayed to determine its melanin content. By comparing their volume-weighted mean nuclear volume and their number-weighted mean nuclear volume, we have found that the nuclei of Cloudman melanoma cells form a fairly homogeneous population. The volume fraction and absolute volume of premelanosomes (Vvpm, cell and Vpm) and mature melanosomes (Vvmm, cell and Vmm) were all found to decrease progressively throughout the period of culture as did the number of premelanosomes (Npm) and mature melanosomes (Nmm). Whilst the volume-weighted mean volume of individual stage I and stage II premelanosomes, (vipm), remained fairly constant at about 10 nm3, the volume of individual stage III and IV mature melanosomes showed significant variation ranging between about 13 nm3 and 32 nm3. The melanin content of the cells decreased progressively over the 4 days of culture. There were, however, considerable variations in both the average melanin content per unit volume of mature melanosomes, in the range 170–600 fg/μm3, and in the melanin content per individual mature melanosome, in the range 3–12 fg. Our findings show that stereological techniques can provide unbiased and sensitive tools for the study of the morphological basis of melanogenesis; their value will become even more evident when they are combined with techniques for the localization of melanogenic enzymes and their substrates