Effects of fluvastatin and coenzyme Q10 on skeletal muscle in normo- and hypercholesterolaemic rats.

Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition.

A comparative study on dyslipidaemia inducing diets in various rat strains.

In our experiments we compared the serum lipoprotein lipid composition of Fischer 344 (F344) and Long-Evans (LE) inbred rats as well as of their hybrid FLF(1) from both sexes after feeding them for 2, 4 and 8 weeks with different diets. The following diets were used: 1) standard diet marked as CRLT/N; 2) diet reach in butter marked as BR; 3) diet containing cholesterol, sodium cholate and methylthiouracil marked as CR; 4) diet marked as BRC, which is the Hartroft-Sós diet modified by our research group consisting of the diets BR and CR. The latter diet was the most effective, because within two weeks the level of serum total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride in the F344 female rats increased 8, 30, 4 and 8 times, respectively. The male rats of the Long-Evans strain showed moderately increased values while the FLF(1) female hybrids derived from the hybridization of LE males and F344 females had values closer to those of the mother strain. Despite the fact that during this time the LDL/HDL ratio increased from 0.06 to 2.97 and the PON-1 activity decreased to one-third, a significant lipid deposition could not be shown in the wall of the abdominal aorta even two months later. Our experimental model is suitable for the chemoprevention of dyslipidaemia or rapid testing of molecules chosen for its treatment.

Lymphoid metastasis of rat My2/De leukemia.

By grafting spontaneous leukemia tumor cells, the myeloid My2/De leukemia rat model was established. Death was caused by impaired functions of heavily infiltrated organs. In vitro culturing of tumor cells, blood and bone marrow counts and cytochemic reactions indicated the leukemic the origin resembling human myeoloblastic leukemia. Metastatic spread was followed after i.v. and i.p. injection, and by implantation of leukemia cells under the renal capsule of rats. Primary tumor and metastasis formation was visualized by (18)FDG or (11)C-methionine administration and MiniPET. The accumulation of radiotracers was measured in different organs and expressed as Differential Absorption Ratios (DARs). Subrenal implantation of My2/De cells resulted in their appearance in other abdominal organs and in parathymic lymph nodes. The release of tumor cells from the primary kidney to the peritoneum was mimicked by the i.p. administration of ink particles. Ink particles deposited in the abdominal organs and in the thoracal lymph nodes, preferentially in parathymic lymph nodes, confirming the notion of lymphatic spread of metastasis.

Changes in superoxide anion production and phagocytosis by circulating neutrophils during tumor progression in a rat model.

The functional state of circulating neutrophils was monitored in a rat model of mesoblastic nephroma during tumor progression. Superoxide anion (O2.-) production in response to PMA and phagocytosis of yeast particles (Saccharomyces cerevisiae) were measured every second day after tumor cell implantation. Both phagocytosis and PMA-induced 02.- generation were found to be enhanced in the first period (on Days 6, 8, and 10), while they became significantly reduced in the advanced stage of cancer (on Days 12, 14, 16, and 18). The suppression of PMNL functions was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. Studies were also carried out on PMNLs isolated from normal rats and the cells were treated with plasma samples obtained from tumor-bearing animals at different stages of nephroma. Incubation of the normal cells with plasmas separated on the 2nd and 8th days of tumor growth influenced neither the 02.- generation nor the phagocytosis. In contrast, plasma preparations obtained on the 14th day significantly inhibited both 02.- production and phagocytosis by normal neutrophils. The alterations in 02'- generation and phagocytosis by PMNLs were observed in close association with tumor growth, thus they could be considered as indicators of tumor progression. However, further studies are required to see whether the granulocyte dysfunctions observed in our animal model could provide additional prognostic information in the case of human malignancies as well as to clarify the origin of inhibitory factor(s) present in the blood of tumorous animals.

Genotoxicity studies on urine and bone marrow samples of rats bearing transplanted nephroma.

It was demonstrated earlier that urine of rats bearing transplanted mesoblastic nephroma had high mutagenic activity in Salmonella typhimurium, which could not be detected in serum samples of the same animals. In this paper, cytogenetic alterations are discussed and the lack of enhanced micronucleus formation in bone marrow of tumorous rats is described. The cytogenetic effect of the hydrophobic (XAD-4) urinary fraction, which has been found to be mutagenic in the TA98 Salmonella strain, was examined in CBA mice. Sister chromatid exchange (SCE) analyses were performed on bone marrow cells of animals treated with single injections of concentrated urine samples. Significant and continuous increases could be detected in the SCE frequencies caused by the urinary concentrates with development of the tumour. Pseudouridine, a suggested urinary tumour marker nucleoside, was also studied for mutagenicity in the Ames Salmonella test. Both derivatives (alpha and beta), however, failed to induce mutations in the TA98/TA100 strains, either with or without metabolic activation. In conclusion, urinary mutagen(s) produced during the renal tumour growth have a spectrum of genotoxicity involving at least two endpoints, but the high pseudouridine excretion may not be responsible for these effects.

Curcumin does not alter the phorbol ester effect on cell-cell transfer of lucifer yellow CH.

Curcumin, the dietary pigment responsible for the yellow color of curry, has been reported to be a potent inhibitor of tumor promotion in mouse epidermis. Since most tumor promoters inhibit cell-cell communication, we have examined the effect of curcumin on the reduction of gap junctional intercellular communication induced by the phorbol ester phorbol-12,13-dibutyrate (PDBu) in BALB/c 3T3 cells. Treatment of cells with 50 microM curcumin slightly inhibited the dye coupling evaluated by intercellular transfer of a fluorescent dye Lucifer Yellow CH; however, lower concentrations of curcumin did not affect the level of intercellular communication. Addition of 200 nM PDBu caused a rapid reduction of dye coupling, which was not altered by either pretreatment or simultaneous curcumin addition.

Urinary and serum mutagenicity studies with rats bearing experimental tumours.

Urine and serum samples of rats bearing three different experimental tumours (hepatocellular carcinoma, myelomonocytic leukemia and mesoblastic nephroma) were investigated for mutagenicity with the Ames Salmonella test. Enhancement of mutagenic activity in TA98 and TA100 was observed only in the case of urine samples obtained from animals bearing nephromas. Mutagenicity increased with increasing time after implantation of tumours. There was no coincidence between urinary and serum mutagenicity under the experimental conditions employed. Further studies are needed to determine the origins, and chemical and genotoxic characteristics of urinary mutagens. In addition, the question as to whether any mutagenic substances can be detected in fractions of plasma/serum should also be experimentally addressed.

Early effects of cyclosporin A on in vivo oncogene expression.

Cyclosporine is a widely used immunosuppressant drug, but development of secondary malignancies has been observed after Cyclosporine containing treatments. In our study we investigated the early effect of Cyclosporin A on oncogene expression. 6, 12, 18 and 24 hours after 80 mg/bwkg Cydosporin A treatment, RNA was isolated from different organs, slot blotted onto Hybond N+ membrane and hybridized with chemiluminescently labelled oncogene probes. As a positive control, rehybridisation with beta-actin probe was used. X-ray films were exposed for 15 minutes, developed, evaluated with densitometer, and the results were expressed as a ratio to the positive control. There was no effect of Cyclosporine A on the expression of c-sis and il-2 receptor genes. The expression of N-ras, c-myc, c-ptc/ret and c-myb oncogenes was elevated in the thymus, lymph nodes, spleen and liver, either transitionally or continuously. We could not detect any elevated gene expressions in the kidney, lung and bone marrow.

The separation of the granulocytes from different rat strains. A comparative study.

Different Percoll density gradients were used to purify granulocytes from Long-Evans, Fischer, Sprague-Dawley, and Fischer/Long-Evans hybrid rats. Three different discontinuous gradient types were developed which permitted the separation of polymorphonuclear leukocytes (PMNLs) from the peripheral blood of the different rat strains. Our purification techniques were compared to each other in terms of purity and yield. Purities of 97.7 +/- 0.6%, 97.0 +/- 1.1%, 96.4 +/- 1.2% and 96.7 +/- 1.1% were achieved for the granulocyte fractions of LE, F344, SD and FL/F1 rats, respectively. The superoxide production of the isolated cells was also investigated and it was established that the granulocytes could be activated by phorbol-12-myristate-13-acetate (PMA).

Studies on acute myelomonocytic leukemia in LBF1 rats.

Granulocytic leukemia was induced in Long-Evans (LE) rats by using the Huggins and Sugiyama method. After serial passage the cells became transformed. The newly transformed cells could be transplanted to LBF1 hybrid rats and observed more readily. A quantity of 10(8) cells/100 g body weight was injected intravenously and after 2-3 weeks myelomonocytic leukemia developed. By examining the bone marrow, spleen and lymph nodes, cytochemical tests verified this transformation. Transplanting 10(2)-10(4) cells under the renal capsule, a quickly growing solid tumor was observed, which caused metastasis to the parathymical lymph nodes and peritoneum. The investigation of oncogene expression for the myc and ras families revealed the presence of myc p62 and ras p21 oncoproteins in the tumor cells by using monoclonal antibodies in immunohistochemical tests. LBF1 rats proved to be good models in obtaining solid tumor growth and myelomonocytic leukemias, equivalent to human M4-M5 type leukemia.

[A gene inhibiting the development of human retinoblastoma: the prototype of tumor suppressor genes]. / Az emberi retinoblastoma kifejlödését gátló gén: a tumor szuppresszor gének prototípusa.

One of the most important progress in cancer research of our time is the discovery of protooncogenes, oncogenes and tumor suppressor genes. The authors review the isolation, of the human retinoblastoma (Rb) susceptibility gene, the analysis of Rb protein and their role in the pathogenesis of retinoblastoma and other tumors.

Tumor cell implantation with the use of Gelaspon gelatin sponge disc.

Myelomonocytic leukemia (My) mesoblastic nephroma (Ne) and hepatocellular carcinoma (He) cells were implanted under the renal capsule of F344, Long-Evans (LE) and BDIX rats. Gelaspon sponge discs were used in the implantation procedure, which were resorbed within a few days. The tumor cells, which were located on the surface of these discs could then attach themselves to the renal capsule and thus grow. There was a correlation between the number of tumor cells and the difference between the two kidney masses. The correlation was linear between 10(4) and 10(6) cells, thus the method proved to be a simple, fast and quantitative model in experimental cancer prevention and therapy.

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats.

Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any well-known organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.

Purification and partial characterization of protein phosphatases from rat thymus.

Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.

N-ras mutation in chemically induced rat brain tumour.

Rat brain tumour was induced by treatment with N-ethyl-nitrosourea. Using Southern blot analysis, restriction fragment length polymorphism of N-ras gene was identified. Comparative studies showed that new restriction site did not occur in the DNA of DMN induced renal and liver tumours. The data suggest that the mutation occurring may be specific to the "target" cell or to the structure of carcinogens.

Plasminogen activator and plasmin-like activities in experimental rat tumours.

Plasminogen activator activity and plasmin-like amidolytic activity were investigated in two experimental rat tumours, using human plasminogen and chromogenic peptide substrate, S-2251. The invasive hepatocarcinoma and non-invasive nephroma were induced with the same chemical carcinogen, dimethylnitrosamine, in F-344 rats and they were continuously transplanted under the renal capsule. While there was no difference in plasmin-like activities of the tumours, the plasminogen activator activity was very low in the nephroma, but high in the hepatocarcinoma. Since the activator activity was completely inhibited by amiloride, it was considered to be of urokinase-type. These results were in accordance with the assumed role of urokinase in the invasion. However, of the respective control organ, kidney was rich in both activities but rat liver contained only very low activities. Therefore the comparison of the plasminogen activator activity of a tumour to the control organ probably does not provide information concerning the malignant transformation as it is suggested in the literature.

The effect of alpha-difluoromethylornithine on ornithine decarboxylase activity in compensatory growth of mouse lung.

After removing the left upper lobe, the remaining lung of mice undergoes compensatory growth. Increased ornithine decarboxylase activity is characteristic of the process. The effect of alpha-difluoromethylornithine was studied on the ornithine decarboxylase (E.C.4.1.1.17.) activity and on the lung growth in mice after lobectomy. Although a single injection of alpha-difluoromethylornithine inhibited ornithine decarboxylase activity, the compensatory growth of lung still occurred in spite of continuous alpha-difluoromethylornithine treatment. This suggests that polyamines, indispensable for compensatory growth, may be supplied by other sources.

Transplantation of chemically induced mouse leukaemia into newborn F344 rats.

Chemically induced lymphoid mouse leukaemia was successfully transplanted into newborn F344 rats. The developing rat-leukaemia contained not only donor cells but donor and recipient cells, or only recipient cells.

Migration and colonization of leukemic lymphoblasts in AKR and HSS inbred mice followed by serial enzyme determinations.

After transplantation of AKR and HSS leukemic cells into AKR and HSS inbred mice as well as into their hybrids, the distribution and proliferation of the cells depend on the host organism. The energy production and glycolytic enzyme activity of the settled cells differ from that of the injected leukemic cells. It may be supposed that either the cell transplantation was carried out with a heterogeneous cell population or the cells of the host organism started to dedifferentiate.

Changes in L-ornithine decarboxylase activity in regenerating lung lobes.

Growth of regenerating lung lobes is preceded by an increase of the activity of L-ornithine decarboxylase (ODC). Mechanical factors play a role in determining both the growth rate and enhancement of activity of ODC in regenerating lung lobes. Hormonal regulation also appears of importance as shown by the increased ODC activity in a transplanted lobe.