Comparison of the effects of tamoxifen and toremifene on rat hepatocarcinogenesis.

The hepatoproliferative and cytochrome P450 enzyme inducing effects of two antiestrogens, tamoxifen and toremifene, were compared in female Sprague-Dawley rats using immunohistochemical staining methods. Equimolar doses of the antiestrogens (tamoxifen 45 mg/kg and toremifene 48 mg/kg) were given by oral administration to 6-week-old rats for 12 months including a 3-month recovery period. Controls received the vehicle carboxymethylcellulose. Altogether 90 rats were used in the study. Five rats per dose group were killed after 14 days, 5 weeks, 3, 6 and 12 months of treatment as well as after the 3-month recovery period. Hepatocellular carcinoma was found in four out of five rats after 12 months of tamoxifen treatment. After the 3-month recovery period all tamoxifen-treated rats had large liver tumors (diameter up to 3 cm). No tumors were observed in toremifene-treated rats. Liver cell proliferation was measured by the index of proliferating cell nuclear antigen (PCNA) expression. Immunohistochemical staining with the placental form of glutathione S-transferase (GST-P) was used as a marker for preneoplastic foci. Cytochrome P450 induction was measured using specific antibodies to isoenzymes. Tamoxifen increased the incidence of GST-P-positive foci significantly by 3 months of treatment but toremifene did not as compared with the controls. Liver cell proliferation increased significantly only in the liver tumors of tamoxifen-treated rats after 12 months of treatment and during the recovery period. Both antiestrogens induced the isoenzymes CYP2B1/2 and CYP3A1 within 14 days although tamoxifen was a more powerful inducer. Immunohistochemistry of rat liver sections showed a centrilobular localization of these induced enzyme proteins. The expression of CYP2B1/2 and 3A1 could also be observed in foci after 3 and 6 months of administration and in liver adenomas and in some carcinomas after 12 months of administration with tamoxifen. The results show that tamoxifen, but not toremifene, has the potential to induce and promote the development of rat hepatocarcinogenesis in this experimental model.

The effect of toremifene on bone and uterine histology and on bone resorption in ovariectomised rats.

The effect of the selective oestrogen receptor modulator, toremifene, to inhibit ovariectomy-induced bone loss was studied in rats. The oral doses were 0.3, 3.0 or 30 mg/kg/day for 2 months. 17beta-oestradiol (5 microg/kg/day, subcutaneously) was used as positive control. One group was also treated with a combination of 17beta-oestradiol (5 microg/kg) and toremifene (3.0 mg/kg). Biochemical markers were urinary hydroxyproline and calcium (adjusted with urinary creatinine levels) and the serum level of pyridinoline cross-linked carboxy terminal telopeptide, a bone specific collagen breakdown product. The femoral and sternal trabecular bone thickness served as histological parameters. Ovarectomy increased the levels of hydroxyproline and pyrodinoline and decreased the trabecular bone thickness compared to the sham-operated control group. This was inhibited by both test compounds but 17beta-oestradiol was more efficient. Toremifene did not reverse the ovariectomy-induced reduction of urinary calcium but inhibited the 17beta-oestradiol-related increase. When administered together with oestradiol, toremifene did not reverse the positive effect of 17beta-oestradiol on bone, however toremifene reversed the oestradiol-related uterothrophic effects. These findings indicate that the antagonistic features of toremifene dominate in the rat uterus the agonistic properties do in the bone.

A two-year dietary carcinogenicity study of the antiestrogen toremifene in Sprague-Dawley rats.

The carcinogenic potential of the nonsteroidal triphenylethylene antiestrogen toremifene (Fareston) was evaluated in a standard 104-week rat dietary carcinogenicity study. The doses were 0, 0.12, 1.2, 5.0 and 12 mg/kg/day and the number of animals 50/sex/dose group. The body weight gain and food consumption were monitored once weekly (study weeks 1-16) or once every four weeks thereafter (study weeks 17-104). Blood samples were taken at weeks 34, 52 and 104 and the plasma concentrations of toremifene, as well as the two main metabolites (deaminohydroxy)toremifene and N-demethyltoremifene, were measured. All doses of toremifene reduced food intake and body weight gain. Toremifene caused a significant reduction in mortality, which was mainly due to reduced incidences of pituitary tumors. This was evident in all dose groups. Drug-related decrease of mammary tumors in females (at all doses) and testicular tumors in male rats (doses > or = 1.2 mg/kg/day) were also evident. The incidence of the preneoplastic foci of basophilic hepatocytes were significantly decreased in treated female groups. Toremifene induced no preneoplastic or neoplastic lesions. Based on histopathology, no obvious toxicity could be observed. Drug-related changes were observed in the genital organs, thyroid, spleen, mammary gland, adrenal, kidney, stomach and lung. These changes were due to hormonal disturbances or as a result of reduced food consumption or reduced incidences of pituitary, mammary or testicular tumors. This study indicates that toremifene is an efficient antiestrogen in long-term treatment, is well tolerated and has no tumorigenic potential in rats.

Tamoxifen induces hepatocellular carcinoma in rat liver: a 1-year study with two antiestrogens.

The effects of equimolar doses of the triphenylethylene antiestrogens tamoxifen and toremifene on female Sprague-Dawley rat liver were studied in a 52-week toxicity study which included a 13-week recovery period. Liver tumors were found in four out of five rats at the highest dose level of tamoxifen (45 mg/kg per day) after 52 weeks of dosing, and these appeared to be hepatocellular carcinomas in three rats. After the 13-week recovery period all surviving rats in the highest tamoxifen dose group had large liver tumors (diameter up to 2 cm) which appeared to be hepatocellular carcinomas in five out of six rats. No tumor was observed in the toremifene-treated rats (48 mg/kg per day) either after 52 weeks of dosing or after the recovery period. Electron microscopic morphometric analysis after 52 weeks of dosing revealed that at the tamoxifen high dose level, the volume densities of the peroxisomes, mitochondria, and residual bodies were elevated in the nonneoplastic hepatocytes of the rats. In the neoplastic hepatocytes of the tamoxifen-treated rats the volume density of nuclei was slightly elevated. The slight proliferation of peroxisomes and mitochondria might be related to tumor development in the tamoxifen treated rats.

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.

Vinblastine-induced autophagic vacuoles in mouse liver and Ehrlich ascites tumor cells as assessed by freeze-fracture electron microscopy.

The structure of the membrane limiting apparently newly formed autophagic vacuoles was studied in vinblastine (VBL) induced autophagocytosis in mouse liver parenchymal cells and in Ehrlich ascites tumor cells. In the VBL-treated cells, the formation of autophagic vacuoles was far greater than in the controls as examined by thin section transmission electron microscopy. In freeze-fracture studies of both VBL-treated and control cells, only the P- and E-fracture faces of the outer limiting membrane of the autophagic vacuoles contained a few intramembrane particles (IMP). Their density appeared however, to be much lower than the IMP density on the P- and E-fracture faces of the endoplasmic reticulum or of the Golgi apparatus. The inner limiting membrane of the autophagic vacuoles was smooth. It is apparent that the membranes of endoplasmic reticulum or Golgi apparatus are not directly involved in autophagic vacuole formation.

beta-Glucuronidase activity in trained red and white skeletal muscle of mice.

We studied the effects of prolonged running exercise (5 days a week, 1.5 h per day at a speed of 17.6 m/min) on the activity of some acid hydrolases (beta-glucuronidase, beta-N-acetylglucosaminidase, acid phosphatase and cathepsin D) and three enzymes of energy metabolism (cytochrome c oxidase, lactate dehydrogenase and creatine kinase) in the distal and in the proximal, the predominantly white and red parts, respectively, of the vastus lateralis-muscle from mice. The acid hydrolase activity levels were 1.24--1.69 higher in untrained red muscle compared to untrained white muscle. The light training applied increased the activity of beta-glucuronidase in both red and white muscle. No other significant training effects were observed in the enzyme activities measured.

Sex differences in renal damage induced in the mouse by Amanita virosa.

The sensitivity of male and female mice to Amanita virosa was compared. Dried, homogenized mushroom was given orally by stomach tubing at doses of 100, 200, 400 and 800 mg dried mushroom/kg body weight. Both in males and in females, the kidneys were the only organs showing macroscopical changes. The dose of 100 mg/kg caused renal damage in females, whereas in males the first signs of kidney damage were seen at the dose of 400 mg/kg. The renal lesions observed in the males were located in the cortex, while in the females they were limited to the outer stripe of the outer medullary zone. Testectomy diminished the nephrotoxicity of A. virosa in male mice and caused changes in the localization of renal lesions.

The effect of furosemide on the renal damage induced by toxic mushroom Cortinarius speciosissimus in the rat.

The effect of furosemide on the renal damage induced by the toxic mushroom Cortinarius speciosissimus was studied in female rats. Furosemide (50 mg/kg s.c.) was given 15 min before the mushroom administration (250 mg dried mushroom/kg body weight p.o.). It was observed that furosemide clearly increased the tubular damage induced by C. speciosissimus. In contrast, furosemide had no effect on the inflammation induced by this toxic mushroom.