Tumor promoters as inhibitors of apoptosis in rat hepatocytes.

Multistage carcinogenesis in rat liver is widely used as an experimental model for the study of the critical events in tumor promotion. After an initial treatment with a genotoxic liver carcinogen ('initiation'), subsequent application of certain non-genotoxic agents can lead to the clonal expansion of putative preneoplastic cells ('promotion'). Obviously, the expansion of these clones is correlated with an increased occurrence of benign and malignant liver tumors at later time points. Since both proliferation and apoptosis were reported to be enhanced in putative preneoplastic liver foci, inhibition of apoptosis was suggested to play a critical role in tumor promotion. In rat hepatocytes in primary culture, the liver tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibited apoptosis initiated by treatment of the cultures with UV irradiation but did not affect apoptosis in non-irradiated cultures. The suppression of apoptosis with TCDD coincided with an attenuated increase of the tumor suppressor protein p53 observed upon UV irradiation. Furthermore, TCDD treatment resulted in a marked hyperphosphorylation of p53. The fact that almost identical concentration-response curves were obtained for the phosphorylation of p53 and the induction of cytochrome P450(CYP)1A-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity indicates that p53 phosphorylation after TCDD treatment is mediated by the aryl hydrocarbon receptor (AhR) signaling cascade. With tumor-promoting 'non-dioxin-like' polychlorinated biphenyls inhibition of UV-induced apoptosis was also observed. A comparative study investigating the effects of various concentrations did not reveal, however, a clear correlation between the suppression of apoptosis and the induction of CYP2B-catalyzed 7-pentoxyresorufin O-dealkylase (PROD) activity. In summary, inhibition of UV-induced apoptosis with liver tumor promoters is observed in rat hepatocytes in culture. Hyperphosphorylation of key proteins of apoptosis including p53 seems to play a role in this effect.

2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses apoptosis and leads to hyperphosphorylation of p53 in rat hepatocytes.

Inhibition of apoptosis of preneoplastic cells is thought to represent a major mechanism of action of tumor promoters. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent promoter of liver carcinogenesis in rodents, suppressed apoptosis in rat hepatocytes pretreated in vitro with an apoptogenic dose of UV light. This effect, which was also observable in DNA fragmentation analysis, coincided with a pronounced inhibition of the p53 increase usually seen after UV irradiation of rat hepatocytes. Interestingly, TCDD also led to a very minor but consistent enhancement of DNA fragmentation and to a slight increase in p53. Furthermore, TCDD resulted in a dose-dependent increase in p53 phosphorylation in intact cells. The concentration-response curves of the effects of TCDD on p53 phosphorylation and aromatic hydrocarbon receptor (AhR)-dependent induction of cytochrome P450 1A1 activity were almost superimposable, suggesting that TCDD induces p53 phosphorylation via an AhR-linked kinase activity. In an extract prepared from rat liver homogenate, 1 nM TCDD also stimulated p53 phosphorylation. Since the tyrosine kinase c-src was previously shown by others to be activated upon binding of TCDD to the AhR, extracts were pretreated with anti-src-antibodies. This treatment almost completely abrogated the effect of TCDD on p53 phosphorylation suggesting a key role for AhR-associated c-src. This mode of action may result in the observed suppression of the p53 response to apoptogenic UV irradiation, and may contribute to the inhibition of apoptosis.

Influence of liver tumor promoters on apoptosis in rat hepatocytes induced by 2-acetylaminofluorene, ultraviolet light, or transforming growth factor beta 1.

DNA damage is recognized widely as a cause of programmed cell death (apoptosis), aimed at eliminating cells bearing genotoxic lesions. Therefore, inhibition of DNA damage-induced apoptosis may play an important role in carcinogenesis and has been suggested as a mechanism of action of tumor-promoting agents. In the present study, the effects of treatment with UV light or the carcinogenic aromatic amine 2-acetylaminofluorene (2-AAF) on apoptosis were studied in rat hepatocytes in primary culture. A significantly increased incidence of apoptotic nuclei, showing condensed or fragmented chromatin visualized with the fluorescent dye Hoechst 33258, was found after each type of treatment. After 48 h, the incidence of apoptosis had returned to the control level. When the liver tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and phenobarbital were added to the medium, apoptosis did not increase in UV- or 2-AAF-treated compared with untreated cultures. Furthermore, TCDD and phenobarbital suppressed internucleosomal DNA fragmentation elicited by UV irradiation. In contrast, the promoters did not suppress apoptosis induced by transforming growth factor beta 1. Immunoprecipitation of the tumor suppressor gene product p53 demonstrated that the increase in p53 observed after UV irradiation was abrogated almost completely by TCDD. Apoptosis induced in rat hepatocytes by DNA-damaging agents such as UV light or 2-AAF is suppressed by TCDD and phenobarbital. Inhibition of apoptosis allowing survival of hepatocytes bearing genotoxic lesions may be crucial for the tumor-promoting action of TCDD and phenobarbital in the liver.

Influence of non-steroidal anti-inflammatory drugs on the binding kinetics of dansylsarcosine to human serum albumin. Stereoselectivity, steric and inductive effects.

The effect of a series of non-steroidal anti-inflammatory drugs (NSAIDs) on the binding kinetics of dansylsarcosine (CAS 72517-44-3, DS), a marker ligand for the benzodiazepine binding site, and human serum albumin (HSA) was studied using the stopped-flow method. Both native (7% glycated) and 25% glycated HSA were used. The binding parameters were determined on the basis of the consecutive model. The DS association rate constant (k2) was 649 +/- 84 s-1 and 375 +/- 13 s-1 for 7% and 25% glycated HSA, respectively. These values were substantially influenced by addition of NSAIDs (molar ratio HSA:NSAID = 2:1), depending on the structure of NSAIDs. The calculated DS dissociation rate constant (k-2) was approximately 20 s-1. this value did not show marked dependence on the degree of glycation or on the presence of NSAIDs at the concentration used. The values were similar to estimates of kd (the displacement rate constant of DS) with the exception of diclofenac (CAS 15307-86-5) where kd was significantly lower, reaching 4.8 +/- 0.4 s-1 and 4.8 +/- 0.6 s-1 vs. k-2 parameters of 14 +/- 2.8 s-1 and 15 +/- 3.7 s-1 for 7% and 25% glycated HSA, respectively. A comparison of the enantiomers R- and S-ibuprofen (CAS 15687-27-1) and the regioisomers fenbufen (CAS 36330-85-5) and ketoprofen (CAS 22071-15-4) showed slight or no stereoslectivity of effects on the DS binding kinetics. However, the binding was influenced by bulk and nature of substituents at the aryl rest of propionic acid. The results obtained for mefenamic acid (CAS 61-68-7) suggest that this NSAID binds to a site of human serum albumin other than site II. Increased concentrations of glycoalbumin, as observed in diabetic patients, are not presumed to have inhibitory effects additional to that of NSAIDs which interact differentially with drugs at site II of HSA.

Effect of free fatty acids on the binding kinetics at the benzodiazepine binding site of glycated human serum albumin.

The effect of free fatty acids (FFA) and non-enzymatic glycation on the binding kinetics of dansylsarcosine (DS) to human serum albumin (HSA) was studied using the stopped-flow technique. The influence of FFA on the binding parameters of 25% glycated HSA depended on the type of fatty acid. The addition of stearic, oleic and linoleic acids in a concentration of 0.3 mmol/l showed no inhibitory effects on the association rate constant (k2) value for DS binding to 25% glycated HSA (k2 without FFA: 385 +/- 10 s-1, k2 with FFA > or = 385 +/- 10 s-1). In contrast, shorter chain fatty acids (hexanoic, octanoic, decanoic, lauric and myristic acids) showed marked inhibitory effects for 0.3 mmol/l FFA (k2 range: 233 +/- 32 to 69 +/- 5 s-1) and for 0.6 mmol/l FFA (k2 range: 125 +/- 3 to 20 +/- 4 s-1). The association rate constant (k2) as well as the affinity constant (KA) of DS were markedly affected by glycation: k2 was 686 +/- 61 s-1 for 7% glycated HSA, 385 +/- 10 s-1 for 25% glycated HSA and 209 +/- 12 s-1 for 50% glycated HSA. KA decreased from 6.1 +/- 2.9 x 10(5) M-1 for 7% glycated HSA, to 5.1 +/- 0.1 x 10(5) M-1 for 25% glycated HSA and to 1.3 +/- 0.6 x 10(5) M-1 for 50% glycated HSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation parameters determine role of GABAB receptors in long-term potentiation.

Blockade of GABAB receptors was reported to improve cognitive performance in mammals. The physiological basis of this effect is poorly understood. We investigated the effect of the GABAB receptor antagonist CGP 35348 on long-term potentiation (LTP) in the CA1 area of the hippocampus in vitro and in vivo. In vitro the effect of CGP 35348 on LTP, induced either by two non-primed tetanic stimulations or by two primed bursts of stimuli, was investigated. In the presence of 1 mM CGP 35348 LTP was significantly facilitated following two non-primed tetanic trains, but was impaired following two primed burst stimulations. In vivo LTP was induced by applying non-primed trains of stimuli of increasing duration to the Schaffer collateral/commissural fibers. The potentiation of the population spike recorded in CA1 was significantly facilitated by CGP 35348 (100 mg/kg i.v.). In conclusion these findings demonstrate that the GABAB antagonist CGP 35348 facilitates LTP in vitro and in vivo if induced by non-primed tetanic stimulation. In vitro, the mode of stimulation determines the effect of the GABAB antagonist on LTP.

Selective determination of non-enzymatic glycosylated serum albumin as a medium term index of diabetic control.

Serum proteins are non-enzymatically glycosylated dependent on the concentration of free glucose and measurements of their concentration are used to control diabetic carbohydrate metabolism. Eight patients with insulin-dependent diabetes mellitus (IDDM) and 8 patients with non-insulin-dependent diabetes mellitus (NIDDM) with glycosylated hemoglobin levels of at least 10.5% were studied during a 6-week period of antidiabetic therapy. Glycosylated serum albumin (GSA) and glycosylated total serum proteins (GSP) were measured weekly using an affinity chromatography procedure. The fructosamine test (FA) and the measurement of mean blood glucose (MBG) were also carried out weekly. Glycosylated hemoglobin and its glucose adduct HbA1c were determined at 14-day intervals (HPLC-method). All measured parameters decreased during the period of the study. The correlation coefficients for the glycosylated proteins versus the MBG determined one week earlier were highest for GSA [IDDM: r(GSA/MBG-1) = 0.726, p < 0.001 for the single values and 0.984, p < 0.001 for the mean values; NIDDM: r (GSA/MBG-1) = 0.636, p < 0.001 for the single values and 0.986, p < 0.001 for the mean values]. The differences between the IDDM and NIDDM group probably occurred because 6 NIDDM patients were taking glibenclamide (7.0-10.5 mg/day) which is known to inhibit the glycosylation reaction of albumin. The fructosamine test is more prone to interferences than the selective determination of GSA. GSA determination therefore, gives precise data in medium term diabetic control.

Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases.

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.

Oxygen-dissociation kinetics in the blood of smokers and non-smokers: interaction between oxygen and carbon monoxide at the hemoglobin molecule.

The importance of smoking as a possible factor in coronary heart disease (CHD) may be related to the effect of carbon monoxide (CO) on oxygen exchange at the hemoglobin molecule (Hb). We examined the kinetics of this process in vitro and ex vivo using a fast-reaction technique (stopped-flow) whereby the dissociation-rate constant of oxygen was determined in the blood of smokers and non-smokers and at fixed HbCO-concentrations in non-smokers blood. In non-smokers, carbon monoxide saturated blood was obtained by gassing with carbon monoxide and mixing the samples with appropriate carbon monoxide free blood to achieve HbCO-concentration in the range of 10-60%. The reaction time course for the oxygen-dissociation was divided into a non-linear-initial phase (loss of the first oxygen molecule) and a subsequent linear phase. The oxygen-dissociation velocity decreased from 96.5 x 10(3) ms-1 to 42.7 x 10(3) ms-1 in the linear phase at pH 7.4 and decreased from 29.2 x 10(3) ms-1 to 20.9 x 10(3) ms-1 at pH 9.2 when the HbCO-concentration was increased to 63%. For the initial phase at pH 7.4, the dissociation velocity decreased depending on the HbCO-concentration. In non-smokers 50% of the bound oxygen was released in 17.5 +/- 2.3 ms (n = 13) whereas in smokers 19.4 +/- 1.8 ms (n = 14) (p less than 0.05) was required.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-protein binding kinetics in patients with type I diabetes.

Sera from 17 patients with Type I diabetes and 19 healthy volunteers have been examined to evaluate whether the kinetics of the binding of drugs to Site II of serum albumin is altered in diabetes. Stopped-flow measurements showed that the association velocity and the affinity constants of the fluorescent marker dansylsarcosine were significantly lower in diabetes (160 s-1 and 2.0 x 10(5) l.mol-1) than in non-diabetics (196 s-1 and 4.0 x 10(5) l.mol-1). The dissociation velocity was not different [20.3 s-1 vs. 19.4 s-1]. Although patients with a reduced albumin concentration were excluded the diabetics had significantly lower concentrations than the healthy volunteers. There was a significant correlation between decreased glycosylation of albumin and increased association velocity. The dissociation velocity constants were correlated with the molar concentration ratio of free fatty acids/human serum albumin. Thus, the extent of glycosylation and the amount of fatty acids bound per mole albumin can both affect the kinetics of drug binding to Site II. The lower affinity in patients with Type I diabetes is due to the increased in the glycoalbumin concentration.

[Non-enzymatic glycation of human serum albumin: influence on thebinding kinetics of the benzodiazepine binding sites]. / Nichtenzymatische Glykierung von Humanserumalbumin: Einfluss auf die Bindungskinetik an der Benzodiazepinbindungsstelle.

Human serum albumin was non-enzymatically glycated in vitro and the glycation rate determined using an affinity chromatography method. The influence of glycation on the binding of the model ligand, dansylsarcosine, at the benzodiazepine binding site was determined with a stopped-flow method. Fluorescence time curves were recorded during the binding process. As the glycation rate increased, the association velocity constant, k2, decreased from 533.3 s-1 (glycated albumin 0.048 of total serum albumin) to 218.1 s-1 (glycated albumin 0.158 of total serum albumin). The affinity constant, KA, showed a corresponding decrease from 7.61 x 10(5) l/mol (fraction of glycated albumin 0.048) to 2.60 x 10(5) l/mol (fraction of glycated albumin 0.158). The dissociation velocity constant, however, increased from 17.3 s-1 (fraction of glycated albumin 0.048) to 19.8 s-1 (fraction of glycated albumin 0.158). The inhibition of binding probably occurs via an allosteric mechanism.

The oxidation of the highly tumorigenic benz[a]anthracene 3,4-dihydrodiol by rat liver dihydrodiol dehydrogenase.

Rat liver dihydrodiol dehydrogenase (DDH, EC 1.3.1.20) has been shown to reduce the mutagenicity of benz[a]anthracene (BA) in the bacterial Ames test. BA-3,4-dihydrodiol is a highly mutagenic and tumorigenic metabolite of BA. In order to test the hypothesis that this dihydrodiol may be a substrate of DDH, we established two novel assay systems for the NADP(+)-dependent oxidation of BA-3,4-dihydrodiol by rat liver DDH, an HPLC-based assay procedure and a radiometric assay with specifically labelled [3,4-3H]-BA-3,4-dihydrodiol as substrate. With the HPLC-based assay, the kinetic constants of the enzymatic catalysis were as follows: Km(app) = 21 microM for BA-3,4-dihydrodiol and Vmax = 20.0 nmol/min.mg enzyme. The reaction product was identified by cochromatography, fluorimetry and mass spectroscopy as BA-3,4-catechol, but interconversions between the catechol and the corresponding o-quinone during the analytical procedures were detected. With the radiolabelled substrate, a linear relationship between substrate concentration and reaction velocity was found. The V/K value for labelled substrate was 0.155 ml/min.mg enzyme and a (V/K)H/(V/K)T kinetic isotope effect of 6.7 was observed. The non-labelled substrate acted as a competitive inhibitor of the enzymatic oxidation of tritiated BA-3,4-dihydrodiol with a Ki value of 56.4 microM. The reaction rates determined in this study suggest an important role of DDH activity in the metabolism of BA.

Kinetics of drug binding to human serum albumin: allosteric and competitive inhibition at the benzodiazepine binding site by free fatty acids of various chain lengths.

The inhibition of dansylsarcosine (DS) binding at the benzodiazepine binding site of human serum albumin has been studied in the presence of saturated and unsaturated free fatty acids (FFA) of various chain lengths (C6-C20, C18:1, C18:2). In order to determine the mechanism of displacement, velocity constants for association (k2) and dissociation (k-2) and binding constants (KA and KA') have been measured using the stopped-flow method. The inhibitory effect of FFA on DS binding kinetics at site II is dependent of their structure. With increasing amounts of FFA the association velocity constant of DS binding decreases from 520 s-1 (fatty acid free albumin) by a factor of 3-10 and affinity decreases according to FFA chain length. Inhibition is strongest in the presence of caprylic, capric and lauric acid (C8-C12) i.e. with more than one mole FFA per mole albumin, DS association could no longer be measured. Short chain caproic and the long chain FFA C14-C20 showed only a less inhibitory effect since in the presence of a twofold excess k2 ranged between 100 and 200 s-1. Dissociation velocity of DS from the benzodiazepine binding site could be measured in relationship to FFA chain length using ibuprofene, another drug binding at site II. Dissociation velocity constants k-2 remained constant up to 2 moles FFA per mole albumin (k-2 = 16-18 s-1). A rise in k-2 to 70 s(-1) was seen, however, when 2-4 moles capric, lauric, myristic and palmitic (C10-C16) acid were bound, whereas no change was observed when increasing concentrations of caproic, caprylic, stearic and arachic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of rat-liver cytosolic epoxide hydrolase.

Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.

[Chemotherapy and enteral nutrition in stomach cancer]. / Chemotherapie und enterale Ernährung beim Magenkarzinom.

Malnutrition is a frequent problem in cancer patients. About 45% of them lose more than 10% of their original weight at the various stages of their disease. The importance of nutritional support was repeatedly pointed out. In our study, 10 patients with metastatic gastrointestinal cancer received a combination treatment of long-term tube feeding with elemental diets and chemotherapy. The initially low Karnofsky index improved significantly. The results of the chemotherapy are comparable to those of 9 international studies between 1976 and 1979, using a comparable therapeutic scheme in patients with initially higher Karnofsky index. Ingestion-dependent abdominal pain disappeared in responders and non-responders during the time of tube feeding.

Time-dependence and differential induction of rat and guinea pig peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, cytosolic and microsomal epoxide hydrolase after treatment with hypolipidemic drugs.

Fischer-344 rats and Hartley guinea pigs received a diet containing 0.01% (w/w), 0.05% (w/w), or 0.25% (w/w) of the hypolipidemic drug fenofibrate. Rats were treated for 4, 7, 14, or 21 days, and a clear dose-dependent and weak time-dependent increase in liver/body weight ratio was observed. The specific activity of peroxisomal beta-oxidation increased linearly with time at all concentrations used. A dose-dependent increase in cEH was observed, but the activity remained constant after treatment for 7 days. Enhancement of palmitoyl-CoA hydrolase was dose-dependent, but was similar at all 4 time points investigated. In contrast to the other enzyme activities, mEH was not or only minimally (less than 1.5-fold) induced. In contrast to the rat, treatment of guinea pigs with fenofibrate for 1 week did not change liver weight or enzyme activities. Prolonged treatment of guinea pigs (4 weeks) with fenofibrate did not result in an increase in enzyme activities. This was also observed with clofibrate whereas tiadenol caused a slight increase in enzyme activities (1.5- to 2.6-fold). In contrast to the guinea pig each of the three hypolipidemic drugs led to an increase in enzyme activities in the rat liver after treatment for 1 week.