Electrochemical treatment (EChT) effects in rat mammary and liver tissue. In vivo optimizing of a dose-planning model for EChT of tumours.

BACKGROUND: A reinvented technique for tumour therapy, electrochemical treatment (EChT), is attracting increasing attention. This study compared results from treatment of liver and mammary tissue focusing on destruction and pH changes in the tissue close to the treatment electrodes. Subsequently, data were compared with a dose-planning model. METHODS: Mammary or liver tissue in 50 adult female Sprague Dawley rats was given EChT with a constant, direct current. The electrodes used were Pt/Ir (9:1) with spherical tips. In situ pH measurements were taken with a micro-combination glass electrode. RESULTS: Spherical lesions were produced in both liver and mammary tissue. No significant difference was detected when comparing the size of the lesions in the two kinds of tissue. Similar pH profiles were obtained in tissue surrounding the electrodes, with pH values changing rapidly from unphysiological to neutral status within the space of a few millimetres. The pH at the border of the macroscopic destruction zone, regardless of tissue type or coulomb dosage, correlated well with specific values (4.5-5.5 at the anode and between 9 and 10 at the cathode). CONCLUSION: The analogous destruction patterns in mammary and liver tissue support the hypothesis that EChT has similar results in at least these two different types of tissue. This implies that the destructive pattern caused by the treatment may be the same also in tumours.

Increased levels of cytosolic thioredoxin reductase activity and mRNA in rat liver nodules.

BACKGROUND/AIMS: Thioredoxin reductase, a redox active enzyme, is induced in several tumors. This study focuses on the presence of and subcellular localisation of thioredoxin reductase in a tumor model where neoplastic lesions are selected by their resistance to the toxic effects of the promotor. METHODS: Liver nodules produced by intermittent feeding of 2-acetylaminofluorene to male Wistar rats were analyzed for thioredoxin reductase (TrxR) activity and mRNA. RESULTS: This activity was increased 3.5-fold in the cytosol but decreased 60% in the mitochondrial fraction compared to the liver of age-matched untreated animals. Only traces of activity were observed in the microsomal, plasma membrane and nuclear fractions from normal liver or nodules. The level of TrxR mRNA was 3-fold higher in nodules than in normal rat liver. Furthermore, the total level of SH groups in homogenates was 2-fold higher in the case of the nodules. CONCLUSIONS: These findings indicate that the thioredoxin system makes an important contribution to the resistant phenotype of the neoplastic liver cell, which conveys a growth advantage of significance for tumor progression.

Reduction of ubiquinone by lipoamide dehydrogenase. An antioxidant regenerating pathway.

Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min(-1) in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mM and was even further increased at 0.5 mM. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP(+) ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.

Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique.

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%). This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.

Electrochemical treatment of tumours.

The electrochemical treatment (EChT) of tumours implies that tumour tissue is treated with a continuous direct current through two or more electrodes placed in or near the tumour. The treatment offers considerable promise of a safe, simple and relatively noninvasive anti-tumour therapy for treatment of localised malignant as well as benign tumours. Although more than 10,000 patients have been treated in China during the past 10 years, EChT has not yet been universally accepted. The reason for this is the lack of essential preclinical studies and controlled clinical trials. Uncertainties regarding the destruction mechanism of EChT also hinder the development of an optimised and reliable dose-planning methodology. This article reviews the collected Chinese and occidental experiences of the electrochemical treatment of tumours, alone and in combination with other therapies. The current knowledge of the destruction mechanism underlying EChT is presented along with different approaches towards a dose planning methodology. In addition, we discuss our view of different important parameters that have to be accounted for, if clinical trials are to be initiated outside of China.

Diet restriction increases ubiquinone contents and inhibits progression of hepatocellular carcinoma in the rat.

BACKGROUND: The aim of the present study was to evaluate the effect of a moderate diet restriction on the progression of preneoplastic foci into hepatocellular carcinomas (HCCs) and whether such an effect was related to altered cell proliferation, apoptosis, and/or tumour contents of lipid-soluble antioxidants. METHODS: Male Wistar rats were exposed to diethylnitrosamine as initiator and 2-acetylaminofluorene plus partial hepatectomy as promoter. Six weeks after initiation the animals were given a diet restricted to 75%-80% of that given to controls until being killed 45 weeks later. Macroscopic liver tumours were histologically classified. In hepatocellular carcinomas the numbers of S-phase (labelling index) and DNA-fragmented (apoptotic index) nuclei were calculated immunohistochemically, and the tumour contents of alpha-tocopherol and ubiquinone were determined. RESULTS: The number of animals with HCC and the number of HCCs per animal were significantly reduced in restricted-diet animals compared with controls. In HCCs the contents of ubiquinone-9 and -10 were significantly increased, labelling indices were enhanced 3-fold, and apoptotic indices 12-fold as a response to food restriction. Neither the size nor the differentiation of HCCs was altered by food restriction. The numbers and areas of preneoplastic foci were similar in restricted-diet animals compared with those of controls. CONCLUSION: Moderate, long-term food restriction inhibits the progression of preneoplastic liver foci into HCC. Possible mechanisms of this inhibition are a shift in the balance between apoptosis and cell division towards cell death and an adaptive response to oxidative stress by increased tumour contents of ubiquinones.

Effects of dietary iron overload on progression in chemical hepatocarcinogenesis.

AIM: The present study was undertaken to investigate possible effects of dietary iron during the progression step in hepatocarcinogenesis. METHODS: Two experiments were performed, in which preneoplastic foci were produced in rat liver using the Solt & Farber protocol, with diethylnitrosamine as initiator and partial hepatectomy + 2-acetylaminofluorene as promoter. Two weeks after promotion, animals were fed 1.25-2.5% dietary carbonyl iron or a control diet until sacrifice. In the first experiment, animals were killed at different time points when they developed an abdominal mass in combination with weight loss. In the second experiment, animals were sacrificed 45 weeks post-promotion. Liver tumours were counted and histologically graded. Tumour levels of ubiquinone-9 and alpha-tocopherol were determined with HPLC, and labelling and apoptotic indices calculated using immunohistochemistry. The number and area of glutathione S-transferase 7,7 (GST-7,7)-positive foci were determined. RESULTS: In experiment number 1, survival and tumour differentiation were similar in iron-treated animals and controls. In the second experiment, iron-treated rats had an increased number of GST-7,7-positive foci compared to controls. Number and size of carcinomas were similar between the groups, whereas tumour differentiation was higher in rats exposed to iron. Cell proliferation, apoptosis and concentrations of alpha-tocopherol in tumours were not altered by iron. The ratio of reduced/oxidized ubiquinone-9 was decreased in tumours from iron-treated animals. CONCLUSION: In this model, dietary iron overload resulted in an increased number of preneoplastic foci but did not enhance the progression of these into hepatocellular carcinomas. Iron decreased the ratio of reduced/oxidized ubiquinone-9 in tumours, indicating that neoplastic liver cells utilize intracellular ubiquinones as a defense mechanism against iron-induced oxidative stress.

Ubiquinone is reduced by lipoamide dehydrogenase and this reaction is potently stimulated by zinc.

Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 microM in the presence and 4 microM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.

Enzymes of the mevalonate pathway in rat liver nodules induced by 2-acetylaminofluorene treatment.

Certain enzymes of the mevalonate pathway have been investigated in persistent liver nodules induced in the rat by 2-acetylaminofluorene. In these nodules the dolichol level was increased 5-fold, the ubiquinone-9 content elevated 6-fold and the amount of cholesterol unchanged. Microsomal beta-hydroxy-beta-methylglutaryl-coenzyme A reductase activity was greatly increased compared to control liver tissue, which was also the case for the cytosolic farnesyl pyrophosphate synthase. A significant elevation of all-transgeranylgeranyl pyrophosphate synthase activity in the cytosol was also observed. The branch-point enzyme of microsomal dolichol synthesis, i.e. cis-prenyltransferase, was decreased in the nodules; whereas the activity of squalene synthase, the terminal regulating enzyme of cholesterol synthesis, remained unchanged. The dolichol species in nodular tissue were redistributed towards the longer chain length species. One factor regulating the chain length of the polyisoprene products formed in vitro was shown to be the ratio of the concentrations of isopentenyl pyrophosphate:farnesyl pyrophosphate employed. Other regulatory factors in the terminal steps of this biosynthetic pathway appear to determine the amounts and nature of the final isoprenoid compounds formed in vivo. In contrast to the microsomal trans-prenyltransferase activity, which was unchanged, the activity of nonaprenyl-4-hydroxybenzoate transferase, an enzyme participating in ubiquinone synthesis, was greatly elevated. The alterations observed in the activities of enzymes in the mevalonate pathway can at least partially explain the increased levels of dolichol and ubiquinone and the unchanged level of cholesterol found in liver nodules. It is reasonable to propose that this modified mevalonate metabolism will render nodular cells resistant to certain toxic factors and prone to cell proliferation.

Estimation of dolichol and cholesterol synthesis in microsomes and peroxisomes isolated from rat liver.

The participation of peroxisomal and microsomal fractions from rat liver in dolichol and cholesterol synthesis was investigated using marker enzymes. Recovery was 8% for peroxisomes and 33% for microsomes, with virtually no cross-contamination between these fractions. Using these data, it was calculated that the peroxisomal branch-point enzyme activities for dolichol and cholesterol biosynthesis, i.e. cis-prenyltransferase and squalene synthase, were 25% and 12%, respectively, of the total homogenate activity. Treatment with mevinolin increased the peroxisomal contribution in the case of both enzymes, to levels almost equal to that of their microsomal counterparts. These results indicate that peroxisomes play a role in the biosynthesis of isoprenoid lipids and that the extent of this participation is increased extensively when peroxisomes are induced by various treatments.

Lipid compositions of intracellular membranes isolated from rat liver nodules in Wistar rats.

The mevalonate pathway gives rise to important end products for the regulation of growth and resistance to oxidative stress and is, consequently, of importance in carcinogenesis. In this study liver nodules were produced in Wistar rats by intermittent feeding with dietary 2-acetylaminofluorene, and the lipid compositions of isolated microsomes, mitochondria, and lysosomes were examined. The phospholipid compositions of these subfractions were unchanged compared to normal hepatic tissue, but the fatty acid patterns were altered, particularly in microsomes. An increase in the content of palmitic acid and a decrease in that of stearic acid were noted. The pattern of fatty acyl moieties on carbon atoms 1 and 2 of the glycerol backbone of phospholipids was unchanged in nodular tissue compared to normal liver. The amount of dolichol was significantly higher in microsomes and mitochondria, but not in lysosomes, and the relative amounts of longer polyisoprenoid compounds were increased in the liver nodules. The relative concentration of esterified dolichol was decreased and an enrichment in saturated fatty acids in this fraction could be observed. The cholesterol concentration was found to be lower in microsomes, but was unchanged in mitochondria and lysosomes, and the normally low concentration of cholesteryl esters was elevated somewhat in microsomes and lysosomes. The ubiquinone content of liver nodular mitochondria was unchanged, but increased 7-fold in microsomes and 2-fold in lysosomes. The alterations found in the lipid composition of liver nodules are significant and have functional implications in many cellular processes of proposed importance for the carcinogenic process, i.e., protein glycosylation cholesterogenesis, regulation of the mevalonate pathway, cellular oxidation-reduction state, and resistance to oxidative stress.

New brain stem and bone marrow abnormalities in victims of sudden infant death syndrome.

The study looked for new abnormalities in 31 victims of sudden infant death syndrome (SIDS). The focus was on respiratory control centers in the brain stem, because some SIDS victims have had abnormalities in respiratory control during sleep. A major respiratory control area (lateral reticular nucleus) of the medulla was hypomyelinated in 9 of the 31 SIDS victims. In a second study, the size of the 12th cranial nerve nucleus and its neuronal composition were analyzed because this nucleus regulates tongue movements, and the tongue has been postulated to help obstruct the airway in some SIDS victims. The 12th nucleus was found to have a neuronal deficit in more than two thirds of the SIDS victims. Finally, the SIDS victims were found to have a normoblastic hyperplasia in their bone marrows, a presumed response to chronic hypoxemia during sleep.

Management of clonidine ingestion in children.

Six cases of toxic ingestion of clonidine hydrochloride are reviewed. Apnea, respiratory depression, and rhythm disturbances were more frequent in our patients than in those previously reported; hypotension and bradycardia occurred at a similar frequency. Satisfactory management consisted of close attention to vital signs and judicious treatment of specific physiologic abnormalities. Atropine effectively corrected bradycardia. Tolazoline was found to be ineffective in reversing symptoms and signs of clonidine overdosage. Hypotension was managed by volume expansion, and if necessary, by a continuous infusion of dopamine. Naloxone, although not used in our patients, may be of both diagnostic and therapeutic value in treating clonidine overdosage.