[Creatine content and lipid analysis in radiosensitive organs after whole body x-irradiation of mice--a contribution to the further development of biological radiation dosimetry]. / Kreatingehalt und Lipidanalyse in strahlenempfindlichen Organen nach Röntgen-Ganz-körperbestrahlung von Mäusen--ein Beitrag zur Weiterentwicklung der biologischen Strahlendosimetrie.

The authors intended to test the suitability of creatine level and/or certain criteria of lipid analysis in radiosensitive organs for an individual (i.e. considering biologic variations of radiosensitivity), qualitative and quantitative demonstration of an irradiation damage. It was found that creatine determination and several criteria of lipid peroxidation are not suitable for a biologic radiation dosimetry due to the absence of organ dependent, radiation dose dependent, and time dependent changes. The reasons are discussed.

[Efficacy of the paramunity inducers PIND-AVI and PIND-ORF as radioprotective agents]. / Untersuchungen über die Wirksamkeit der Paramunitätsinducer PIND-AVI und PIND-ORF als Strahlenschutzsubstanzen.

A significant reduction of mortality after lethal irradiation (7,8 and 9 Gy X-ray total-body irradiation) was achieved by continuous therapeutic subcutaneous application of the biologic inducers PIND-AVI and PIND-ORF. This was obtained by a stimulation of the investigated spleen parameters and a stimulation of leucocytes and phagocytosis. The inducers had no significant influence on the radiogenic reduction of blood cells and bone marrow cells, of the relative spleen and thymus weight and of the DNA and protein level of spleen and thymus. The regeneration of blood leucocytes (preponderantly by PIND-AVI) and of the spleen (preponderantly by PIND-ORF) was accelerated, but not the thymus regeneration which was already rather low. The leucocyte phagocytosis which increased generally after irradiation was markedly stimulated by paramunization. This stimulation was also observed after a latent time in animals submitted to sham irradiation. Its temporal appearance and its quantitative and qualitative properties corresponded to the effects of inducers after X-ray irradiation. Both inducers were found to be harmless in all experiments.

Essential role of sodium and chloride for theophylline-induced choleresis in the isolated perfused rat liver.

Active secretion of electrolytes by hepatocytes is believed to be responsible for bile acid-independent canalicular bile flow (BAICF). Theophylline, which enhances BAICF, has been shown to enhance electrogenic Cl- secretion in a number of other epithelia. Such transport is dependent on Na+ and Cl-. Thus, the mechanism of theophylline choleresis may also involve stimulation of electrogenic Cl- secretion of the liver. This hypothesis was tested by studying the effect of ion substitution on theophylline choleresis in isolated perfused rat livers. Addition of theophylline (0.1 mmol) and dibutyryl cAMP (0.05 mmol) to 100 ml perfusate, in a single dose, increased bile flow and biliary secretion of Na+ and Cl- reversibly. These effects of theophylline were virtually abolished when perfusate Na+ (146 mM) was replaced by Li+ (146 mM) or choline+ (120 mM), and when Cl- (127 mM) was replaced by 120 mM NO-3, acetate- or isethionate-. Since even the permeable ions like Li+ and NO-3 could not substitute for Na+ and Cl-, these results show that the effect of theophylline on BAICF is specifically dependent on the presence of Na+ and Cl- in the perfusate. We propose, by analogy to other epithelia, that an electrogenic Cl- secretion mechanism is present in the liver. Theophylline, acting via cAMP, stimulates this transport process, thereby enhancing BAICF.

Sodium and chloride dependency of dibucaine- and procaine-induced choleresis in isolated perfused rat livers.

The effect of local anesthetics, dibucaine and procaine, on hepatic bile formation was studied in the isolated perfused rat liver. Perfusate Na+ and Cl- were replaced by other ions to define the possible mechanism of action. A single dose (50 mumol) of dibucaine produced an initial cholestasis followed by choleresis. Whereas dibucaine produced only choleresis at a lower dose (10 mumol), only the cholestatic effect was seen at a higher dose (100 mumol). Procaine, on the other hand, produced only choleresis at all doses (1, 10 and 100 mumol); this choleresis was associated with biliary secretion of procaine and its metabolites. Neither dibucaine nor procaine affected the low endogenous bile acid secretion in these studies. The diffusion permeability coefficient of [carboxy-14C]inulin was not altered significantly by dibucaine and procaine, suggesting no significant alteration of biliary permeability. Biliary secretion of Na+ or Cl- declined during cholestasis and increased during choleresis. The initial cholestatic effect of dibucaine was still present when perfusates Na+ and Cl- were replaced by permeable Li+ or NO3-, but declined when Cl- was replaced by relatively impermeable isethionate, suggesting a nonspecific effect. The choleretic effect of both dibucaine and procaine, however, declined significantly when Na+ or Cl- was replaced by Li+, NO3- or isethionate-. These ion-substitutions did not affect significantly the biliary secretion of procaine and its metabolites. The ability to induce biliary secretion of Na+ and Cl- also decreased when Cl- was replaced by NO3- or isethionate and when Na+ was replaced by Li+, respectively. These results suggest that a part of the choleretic effect of both dibucaine and procaine is specifically dependent on Na+ and Cl-. This fraction is thus unlikely to be due to the osmotic effect of the secreted drug. Further studies showed that dibucaine inhibited Na+-dependent hepatic uptake of taurocholate, suggesting possible interference with other Na+-dependent transport processes. It is proposed that although a part of the choleresis is due to the osmotic effect of the secreted drug, the specific dependency of a portion of the choleretic effect on Na+ and Cl- is due to inhibition of Na+-coupled Cl- reabsorption from the canaliculi.

Therapeutic doses of erythromycin esteolate is not cholestatic in rats in vivo.

The effect of erythromycin esteolate (EE) on bile flow and bile acid secretion was studied in male Wistar rats in vivo. Daily oral treatment with a dose of up to 100 mg/kg for 1 week increased the bile flow and the bile acid secretion. Increasing the days of treatment to 4 weeks with a dose of 20 mg/kg did not alter the measured parameters significantly. Acute intravenous injection of erythromycin lactobionate (50 mg/kg) also increased bile flow and biliary bile acid secretion temporarily. The increase in bile flow may partly be due to the osmotic effect of the drug and its metabolites in bile. Since EE failed to produce cholestasis in the range of therapeutic doses, rats do not seem to be a suitable experimental model for studying EE-cholestasis.

Role of inorganic electrolytes in bile acid-independent canalicular bile formation.

Ion-replacement studies were carried out in the isolated perfused rat liver to obtain insight into the role played by inorganic electrolytes in bile acid-independent canalicular bile flow (BAICF). The BAICF decreased significantly when Na+ (146 mM) was replaced by 120 mM K+, Rb+, Cs+, or choline and when Cl- (127 mM) was replaced by 120 mM acetate or isethionate; there was no reduction in BAICF when Na+ was replaced by Li+ (146 mM) and Cl- by NO-3. K+, Rb+, and Cs+, however, also caused a simultaneous decline in the perfusion rate. The BAICF decreased by 50% when HCO-3 was replaced by equimolar tricine; under this condition replacement of Cl- by NO-3, but not Na+ by Li+, decreased BAICF by 45%. Thus the hepatic transport of Cl- cannot be explained by simple diffusion only, and a special mechanism, probably Na+-coupled Cl- transport, may contribute about 30% of the BAICF. With Li+ replacing Na+ in the medium, the intracellular concentration of Li+ in isolated rat hepatocytes was less than that calculated for electrochemical equilibrium and was increased by 2 mM KCN, indicating active extrusion of this ion. Li+ was unable to activate Mg2+-ATPase of isolated rat liver plasma membranes, and 1 mM ouabain did not affect the Li+ distribution. These results suggest the potential importance of ion pumps other than Na+-K+-ATPase in BAICF.

Importance of solvent drag and diffusion in bile acid-dependent bile formation: ion substitution studies in isolated perfused rat liver.

Ion substitution studies were carried out in the isolated perfused rat liver to define the importance of solvent drag and diffusion in bile acid-dependent bile formation. Two different methods, namely single injection (20 mu moles) and continuous infusions at 0.4, 0.8, 1.2, and 1.6 mu moles per min taurocholate (TC), were used to determine the bile acid-dependent bile flow (BADF). Both methods gave essentially the same results. Replacement of Na+ (146mM) by 120 or 146 mM Li+ and Cl-(127mM) by 120 mM NO3- increased BADF significantly. On the other hand, replacement of Na+ by 120 mM choline and Cl- y 120 mM isethionate decreased the BADF. The osmolarity of TC solution was not different when Na+ was replaced by 120 mM Li+ or choline and TC did not affect the osmotic activity of NaCl, and choline-Cl differently. Thus, the observed effect of Na+ replacement on BADF is not due to any change in the osmotic activity of the secreted TC. Substitution of HCO3- by equimolar tricine also decreased BADF. Under this condition, BADF increased when NaCl was replaced by equimolar NaNO3. Thus, HCO3- does not seem to be essential for TC choleresis. Since Li+ and NO3- are more permeable, and choline and isethionate are less permeable than Na+ and Cl-, respectively, these results suggest that the BADF is dependent on the permeability of the substituting cations and anions and thus support the hypothesis that solvent drag and diffusion play an important role in BADF.

Stereospecific reduction of 3- and 7-oxo groups of oxocholanic acids in isolated perfused rat liver.

The metabolism of 3- and 7-oxo groups of oxocholanic acids was studied in isolated perfused rat liver. The metabolites in bile were determined enzymatically using 3 alpha- and 7 alpha-hydroxysteroid dehydrogenases. The 3-oxo group of all the oxocholanic acids tested (dehydrocholate, glycodehydrocholate, taurodehydrocholate, 3,7-dioxocholanate, 3,12-dioxocholanate and tauro-7,12-dihydroxy-3-oxocholanate) was reduced stereospecifically to 3 alpha-hydroxy metabolites. On the other hand the 7-oxo group was excreted partially unchanged (30% of the dose) and partially as 7 alpha-hydroxy metabolites (6-10% of the dose). The remainder of the 7-oxo group was concluded to have been reduced to 7 beta-hydroxy metabolites. These results indicate that the 7-oxo group of oxocholanic acids is reduced predominantly to 7 beta-hydroxy metabolites in rats rather than to 7 alpha-hydroxy metabolites as found in man.

Age-dependence of molecular and functional changes in biological membrane properties.

Some general aspects including results on the possible mechanisms of membrane ageing are reviewed. The liquid-crystalline fluid state of a biological membrane is an essential condition for maintenance of different membrane functions. The liquid-crystalline state of different plasma membranes changes with age of the organism. The degree of unsaturated fatty acids decreases and the content of cholesterol increases during ageing. It could be shown that superoxide radicals originate from minor side-reactions of oxidoreductase enzymes. Ageing increases the amount of superoxide radicals. A small amount of radicals escape quenching by superoxide dismutase. The formation of radicals leads to degradation of membrane lipids. The age-dependent changes in membrane lipid composition influence respiratory activity in rat heart mitochondria of old animals. Rat liver plasma membrane lipids also show a decrease in membrane fluidity which results in a change in transport parameters of cholic acid and thymidine. The change in age-dependent lipid-protein interactions was demonstrated by spin-label measurements in model membranes. The results demonstrated that peroxidative break-down of lipids is an ongoing post-transcriptional process of ageing. The possible role of protective repair mechanisms is discussed.

Effect of local anesthetics on cellular respiration and thymidine transport in hepatocytes from young and old rats.

The effect of local anesthetics (procaine, nicotinoyl-procaine, tetracaine, and dibucaine) on thymidine uptake and cellular respiration was investigated in hepatocytes from 3- and 24-month-old rats. All local anesthetics inhibited the "high-affinity" as well as the "low-affinity" thymidine transport system in a non-competitive manner immediately upon addition. Nicotinoyl-procaine, tetracaine, and dibucaine showed a similar inhibition profile with an inhibition of 10--15% at 50 mumol/l, or of more than 60% at 1 mmol/l. The less-lipophilic procaine showed a distinctly lower inhibition (10% at 1 mmol/l). The inhibitory effect was reversible and not dependent on Ca2+. All local anesthetics exerted identical effects in hepatocytes from young and old rats. Nicotinoyl-procaine and tetracaine inhibited cellular respiration in young and old rats up to a maximum of 50%. Procaine did not reduce O2 consumption below 1 mmol/l. This inhibition appeared also immediately upon addition, was not reversible, and not dependent on Ca2+. It is concluded that local anesthetics impair quite different biological processes like thymidine transport and cellular respiration in hepatocytes from young and old animals even at concentrations below 100 mumol/l. The magnitude of inhibition was correlated to the lipid solubility of the local anesthetics. The mechanism seemed to be different to the anesthetic action and it is supposed that it is a direct hydrophobic interaction with membrane proteins. Thus the local anesthetic-induced increase in membrane fluidity could not improve the age-dependent impairment of thymidine transport. The reduction of respiration is considered to be due to reduced O2 diffusion. This inhibition is in striking contrast to previously observed stimulatory effects which in part lead to the use of local anesthetics in geriatrics.

Altered hepatobiliary transport of taurocholic acid in aged rats.

Hepatobiliary transport of taurocholic acid was studied in adult (3 months) and old (2 years) rats using an isolated perfused rat liver technique in order to determine the effect of age on hepatic uptake and secretion of bile acids simultaneously. The results were analyzed using a steady-state compartmental model to estimate the uptake and secretion of taurocholic acid. Hepatic secretion was decreased to a greater extent than the uptake in old rats. These changes in transport activities were associated with increases in perfusate and liver bile acid pool sizes. These results can explain the decrease in total pool size and synthesis rate of bile acids observed previously in old rats using in vivo studies. It has been suggested that the age-dependent decrease in bile acid transport capacity of the liver is secondary to the altered lipid composition of the liver plasma membranes of old rats.

Thymidine transport in hepatocytes. An assay for hepatotoxicity.

Thymidine transport and phosphorylation were investigated in isolated rat hepatocytes and AS 30 D hepatoma cells. In contrast to hepatoma cells, hepatocytes exhibited a minimum of thymidine phosphorylation due to a 100-fold smaller thymidine kinase activity. In hepatocytes thymidine is transported by two transport systems: a specific concentrative "high affinity" system and an unspecific non-concentrative "low affinity" system. In hepatoma cells only the "low affinity" system could be detected. A single dose of 20 or 50 mg diethylnitrosamine/kg body weight induced in hepatocytes a remakable increas of thymidine kinase activity and a decrease of the transport by the "high affinity" system. Thymidine transport and phosphorylation by hepatocytes are considered to be sensitive markers for early recognition of toxin-induced liver regeneration.

Responses of mitochondrial superoxide dismutase, catalase and glutathione peroxidase activities to aging.

The previous observation (Eur. J. Biochem., 82 (1978) 563--567) that age-dependent accumulation of lipid peroxides follows as a consequence of increased radical formation in mitochondria has prompted an examination of the response of a set of protective enzymes to the above situation. Levels of mitochondrial catalase activity as well as selenium-dependent glutathione peroxidase activity were found to be increased with age, while superoxide dismutase activity remained unchanged. No selenium-independent glutathione peroxidase activity could be detected either in preparations from young 3-month-old controls or in preparations from 2-year-old rats. Both the relatively high and unchanged levels of reduced glutathione and kinetic considerations suggest that glutathione peroxidase is preferentially involved in lipid peroxide metabolism, while catalase predominantly metabolizes mitochondrial H2O2.