Pirkko Hirsimäki

Toxicity of Antiestrogens

The object of this article is to review briefly the preclinical and clinical safety of some antiestrogens. Tamoxifen, toremifene, droloxifene, and idoxifene are polyphenylethylene antiestrogens, whereas the pure antiestrogen, ICI 182,780 or faslodex, as well as raloxifene, is of a different structure. Tamoxifen has been shown to be genotoxic in several studies. It induces unscheduled DNA synthesis in rat hepatocytes and micronuclei in MCL‐5 a cells in vitro. Tamoxifen also induces aneuploidy in rat liver in vivo and chromosome aberrations and micronuclei in mouse bone marrow. Toremifene has also shown to be genotoxic, but to a far lower extent, by inducing micronuclei in MCL‐5 a cells in vitro and by inducing aneuploidy in rat liver in vivo. Tamoxifen has been shown to be hepatocarcinogenic in the rat in at least four independent long‐term studies. The initiation of tumors in the rat is the result of metabolic activation by cytochrome P450 isoenzymes to an electrophile(s) that binds irreversibly to DNA. The other antiestrogens have not been shown to be carcinogenic in rodents. In several independent clinical studies, the risk of endometrial cancer has increased among tamoxifen‐treated women. After reviewing the available data, the International Agency for Research on Cancer concluded that there was sufficient evidence to show that tamoxifen is a class I human carcinogen. The increased risk for endometrial cancer occurs predominantly among women who are 50 years old or older and who have been treated with tamoxifen. It is not yet clear whether the uterine tumor formation is a result of genetic mechanisms, analogous to those seen in the rat liver or due to the estrogen agonist action of tamoxifen. However, the other antiestrogens with a more or less similar intrinsic estrogenic potential have not been shown to be carcinogenic in humans.

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

SummaryMorphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.

Effects of griseofulvin and nocodazole on the accumulation of autophagic vacuoles in Ehrlich ascites tumor cells.

Accumulation of autophagic vacuoles (AVs) is a phenomenon observed in various cells treated with the microtubule inhibitor vinblastine. In order to test whether the accumulation of AVs is a result of retarded fusion of autophagosomes and lysosomes an investigation was carried out to ascertain whether other antimicrotubular drugs, e.g., nocodazole and griseofulvin, also induce accumulation of AVs. Ehrlich ascites tumor cells were incubated with nocodazole (20 micrograms/ml) or griseofulvin (50 micrograms/ml). Morphometric analyses were performed after incubation periods of 3, 30, 60, and 120 min. The volume densities of autophagic vacuoles did not differ significantly from the control values after the various incubation periods tested. It was concluded that intact microtubules are not needed in the fusion of autophagosomes and lysosomes and that vinblastine accelerates the rate of AV formation.

Studies on the subcellular pathophysiology of sublethal chronic cell injury.

Summary This paper summarizes some of the important subcellular events occurring after chronic sublethal cell injury. Chronic cell injury is defined as the result of injurious stimuli which permit cell survival though in altered steady states for protracted periods of time. The importance of ultrastructural and biochemical studies of these phenomena is emphasized. Among the phenomena discussed are alterations in lysosomes, cellular hypertrophy, fatty metamorphosis, alterations in microfilaments and microtubules, alterations in mechanisms of transcription and replication, disturbances in the cell surface and transport across the cell membrane, and alterations in intracellular transport.

Studies on vinblastine-induced autophagocytosis in mouse liver

SummaryThe microtubule inhibitor vinblastine (25 mg/kg, i.p.) induces autophagocytosis in mouse hepatocytes. The formation of autophagic vacuoles, their contents, and other cellular changes after vinblastine injection in hepatocytes, were studied by light and electron microscopic morphometric analysis. The volume density of autophagic vacuoles increased significiantly during the experimental period (24 h). This increase was due to the significant increase in their number, which was approximately 5-fold 4 h, 12 h and 24 h after vinblastine injection. The mean volume of the autophagic vacuoles increased significantly 1 h after vinblastine injection, at which time the formation of new autophagic vacuoles was at its greatest. There was an accumulation of single membrane-limited, obviously older autophagic vacuoles in the cytoplasm. Their volume density was at its maximum 12 h after injection, suggesting a retarded turnover of autophagic vacuoles. The segregation of cytoplasmic components into autophagic vacuoles may not be selective after vinblastine injection.The injurious effects of vinblastine were evident both in light and electron microscopic studies. In the parenchymal cells the Golgi cisternae were dilated and disorganized and the volume density of the Golgi apparatus was significantly decreased 12 h after vinblastine injection. The volume density of lysosomes was increased during the 12 h after vinblastine injection. Vesicles containing very low density lipoprotein particles accumulated in the cytoplasm so that their volume density was significantly increased during the entire experimental period. Vinblastine apparently interfered with the transport and secretion of the very low density lipoproteins from the parenchymal cells.

Filipin labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

SummaryCholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, doublemembrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazolebuffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic vacuoles were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.

Vinblastine-induced autophagocytosis: The effect of disorganization of microfilaments by cytochalasin B

The effects of disorganization of cellular microfilaments by cytochalasin B on vinblastine-induced autophagocytosis was studied in Ehrlich ascites tumor cells in vitro. Incubation with vinblastine induced a formation of autophagic vacuoles in the cytoplasm. The disorganization of microfilaments by cytochalasin B failed to inhibit vinblastine-induced autophagocytosis. Incubation with cytochalasin B alone induced a rapid formation of blebs on the cell surface. These contained cytoplasmic organelles and were connected by a narrow shaft to the main part of the cell. Thin subcortical microfilaments seen in the control cell cytoplasm were apparently relocated after cytochalasin B treatment and formed amorphous masses deeper in the cytoplasm. Vinblastine did not affect the formation of blebs after cytochalasin B treatment.