Biomedical subjects

F Waechter

Publications and source records attributed to F Waechter.

At least 37 records · Page 2Linked to original sources

PubMed1988

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation.

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.

Animals↗

PubMed1988

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice.

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.

Animals↗

PubMed1988

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor.

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.

Animals↗

PubMed1988

Stimulation of DNA synthesis but not of peroxisomal beta-oxidation by nafenopin in primary cultures of marmoset hepatocytes.

The effects of the peroxisome proliferator nafenopin upon primary cultures of marmoset hepatocytes have been investigated and compared to those on cultured rat hepatocytes. Nafenopin did not induce peroxisomal beta-oxidation or peroxisome proliferation but did induce replicative DNA synthesis. These findings demonstrate that peroxisome proliferation and mitogenicity are two independent properties of nafenopin and question the widely held view that primates are generally insensitive to the effects of peroxisome proliferators.

Animals↗

PubMed1987

Investigations on the mechanism of liver tumour induction by peroxisome proliferators.

Further understanding of the mechanism by which peroxisome proliferators induce liver tumours is essential to assessing the risks of such compounds to exposed humans. To this end the effects of nafenopin upon the liver have been investigated. Nafenopin was shown to induce certain drug metabolising enzymes, but sub-cellular fractions from induced animals did not form reactive metabolites which could be detected as mutagens. Nafenopin treatment slightly increased the rate of alkaline elution of hepatic nuclear DNA from polycarbonate filters. However, simultaneous administration of sodium glycolate to stimulate H2O2 production or pyrazole to inhibit catalase activity had no further effects. These findings demonstrate that nafenopin is not activated to a mutagen and argue against the hypothesis that indirect DNA damage as a result of excess H2O2 production is responsible for tumour induction.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1987

Long-term maintenance of hepatocytes in primary culture in the presence of DMSO: further characterization and effect of nafenopin, a peroxisome proliferator.

The addition of 2% dimethyl sulfoxide to adult rat hepatocytes cultured in a chemically defined medium at Day 1 after cell plating resulted in maintenance of the cytochrome P-450 content and the cyanide-insensitive palmitoyl-CoA beta-oxidation activity at 66 and 70% of the initial Day 1 values. The addition of phenobarbital, 3-methylcholanthrene, or nafenopin from Day 3 to Day 6 increased the contents of cytochrome P-450 to 128, 239, and 251%, respectively, compared to untreated controls at Day 3. In addition, nafenopin also caused a pronounced and time-dependent increase in palmitoyl-CoA beta-oxidation activity but was found to have only a weak stimulating effect on replicative DNA synthesis (2-fold) when compared to that of epidermal growth factor (6.5-fold). In the presence of dimethyl sulfoxide the hepatocyte cultures could be kept alive for more than 1 month. Exposure of such cultures to nafenopin from Day 1 do Day 37 resulted in survival which was even better than that of their untreated counterparts. This effect was accompanied by the appearance of abundant endoplasmic reticulum membranes and an increased number of peroxisomes.

Animals↗

PubMed1986

Species differences in the covalent binding of p-chloro-o-toluidine to DNA.

The covalent binding of p-chloro-o-toluidine to hepatic macromolecules was assessed in rats and mice. At all timepoints investigated covalent binding to DNA was most marked in mice, whilst binding to proteins was more pronounced in rats. Two major hydrophobic DNA-adducts were formed in both species. One of these was formed to a much greater extent (6-30 fold) in mice. Thus, species differences in the metabolism of p-chloro-o-toluidine could account for the fact that mice are more susceptible to the carcinogenic effects of this compound.

Animals↗

PubMed1986

Species differences in the toxicity of p-chloro-o-toluidine to rats and mice. Covalent binding to hepatic macromolecules and hepatic non-parenchymal cell DNA and an investigation of effects upon the incorporation of [3H] thymidine into capillary endothelial cells.

The interaction of p-[14C] chloro-o-toluidine with hepatic macromolecules of rats and mice has been investigated. At all time points after single administration the extent of binding decreased in the order protein greater than RNA greater than DNA in both species. The level of binding to mouse liver DNA was greater than that to rat liver DNA after both single and repeated administration. In vitro studies showed that mouse liver fractions catalysed the binding of p-chloro-o-toluidine to calf thymus DNA more readily than rat liver fractions. Conversely, binding to protein and RNA was more marked in the rat than in the mouse. Species differences in DNA repair rates were not observed. The results failed to demonstrate a preferential persistence of binding to mouse liver nonparenchymal cell DNA. Autoradiographic determinations did not demonstrate any effect of p-chloro-o-toluidine upon the incorporation of [3H] thymidine into subcutaneous capillary endothelial cells. The results suggest that different reactive metabolites are responsible for binding to DNA and protein, and that the pattern of reactive metabolites formed from p-chloro-o-toluidine in the mouse differs from that formed in rats.

Animals↗

PubMed1986

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver.

Pyruvate kinase (PK) isoenzymes, rate limiting for the last steps of glycolysis, were studied in normal rat liver, putative preneoplastic foci, neoplastic nodules and hepatocellular carcinoma. These lesions were produced by an initiation-promotion protocol: treatment with a single dose of N-nitrosomorpholine (NNM) was followed by feeding diets containing phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistry on histological sections with antibodies specifically directed against the L and M2 isoenzymes, (ii) by electrophoretic separation of isoenzymes in homogenates from liver and larger tumors, and (iii) by electrophoretic separation of isoenzymes in parenchymal and stromal cells isolated from liver and tumors. Immunocytochemistry showed decreases of L-PK (L-PK-) in hepatocytes of most of the foci, nodules and carcinomas. Most L-PK- foci showed increases in gamma-glutamyltransferase (gamma-GT) and epoxide hydrolase (EH). PB or alpha-HCH treatment further decreased expression of L-PK in foci, but not in normal liver. Cells and foci with enhanced L-PK (L-PK+) were also found after carcinogen treatment. These did not show increases of gamma-GT or EH or any distinct morphological alterations with the exception of some which were basophilic ('tigroid') in H and E stained sections. No L-PK+ tumors were found. We could not demonstrate the M2-type PK in parenchymal cells of liver or any of the lesions described above. This isoenzyme was restricted to stromal cells in normal rat liver and in all stages of carcinogenesis as shown by immunohistology and by electrophoresis of preparations from isolated cell populations. However, stromal cells from hepatocellular carcinomas exhibited a 3-fold increase of M2-PK compared with stromal cells from normal liver. These results do not support an isoenzyme shift from L to M2-PK in the course of malignant transformation of hepatocytes as suggested previously.

Animals↗

PubMed1985

Induction of hepatic drug-metabolising enzymes following treatment of rats and mice with chlordimeform.

We investigated the effects of the formamidine insecticide chlordimeform upon the activities of various hepatic drug metabolising enzymes in rats and mice. Chlordimeform treatment induced several enzyme activities. However, the extent of induction depended upon the activity studied, the sex of the animal and the species selected. Microsomal cytochrome P-450 content was elevated in both male and female rats and mice. Ethoxycoumarin O-deethylase activity was induced in male and female rats but not in mice, whilst ethylmorphine N-demethylase activity was elevated in mice, but not in rats. Benzo(a)pyrene hydroxylase activity was increased in female rats and mice, but not in males. UDP-glucuronyl transferase, glutathione S-transferase and microsomal epoxide hydrolase were induced in a dose-dependent manner in male rats, and female rats and mice, but not in male mice.

Amidines↗

PubMed1985

Arene imines, a new class of exceptionally potent mutagens in bacterial and mammalian cells.

K-region aziridines of polycyclic aromatic hydrocarbons reverted Salmonella typhimurium his- (TA100, TA98) and Escherichia coli trp- strains (WP2 uvrA), without requiring activation by mammalian enzymes. The number of revertants induced per nmol in S. typhimurium TA 100, the most responsive strain, variea from 6 to 10,000 for the seven monoaziridines and the two bisaziridines tested. Interestingly, the mutagenic potencies (y) of the monoaziridines were closely related (r = 0.984) with those of the corresponding epoxide analogues (x) by the equation y = 19.6 X0.97, i.e., the aziridines were about 20-fold stronger mutagens than were the epoxides. One of the aziridines, benzo(a)pyrene (BP)-4,5-imine, was investigated in several additional mutagenicity test systems: toxicity in DNA repair-deficient (rec-) and -proficient (rec+) Bacillus subtilis strains; induction of 6-thioguanine resistance in V79 Chinese hamster cells; and induction of sister chromatid exchanges in cultured human fibroblasts. In all systems, BP-4,5-imine was much more active than the epoxide analogue, BP-4,5-oxide. The difference in activity was particularly large in the two test systems with mammalian target cells in which several hundredfold higher concentrations of the epoxide had to be used in order to elicit equipotent effects. Even r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP, which is one of the most potent mutagens known for V79 cells, was less active in the mammalian cells than was BP-4,5-imine. One reason that arene imines are such potent mutagens may be that they are poorly detoxified. Addition of highly purified microsomal epoxide hydrolase, which strongly reduced the mutagenicity of BP-4,5-oxide and benz(a)anthracene-5,6-oxide in S. typhimurium, had no effect on the mutagenicity of the corresponding aziridines. Furthermore, while benz(a)anthracene-5,6-oxide was inactivated by highly purified cytosolic epoxide hydrolase, benz(a)anthracene-5,6-imine was not inactivated. It is noteworthy that the arene imines are isomeric with and structurally closely related to aromatic amines. Some aziridines derived from nonaromatic structures (ethylene imines) have been reported as metabolites of xenobiotics; others are used as chemotherapeutics. At present, however, the results are mainly of theoretical interest in that a new type of arene derivatives with exceptionally potent, probably ultimate, mutagenicity was discovered and may be exploited for the study of mechanisms of chemical carcinogenesis.

Animals↗

PubMed1984

Induction of cytosolic and microsomal epoxide hydrolases by the hypolipidaemic compound nafenopin in the mouse liver.

The repeated oral administration of nafenopin, a hypolipidaemic compound, at a dose of 100 mg/kg to male C57BL/6, DBA/2, Balb c and C3H mice caused an increase in the specific activity of liver cytosolic epoxide hydrolase, the activity of microsomal epoxide hydrolase was also increased in all except the C3H mice. The dose dependence and the specificity of this induction was investigated in male DBA/2 mice. In the range of 10-200 mg/kg nafenopin the induction of the two hydrolase activities was found to increase with increasing doses of the test compound. Two other cytosolic enzyme activities, lactate dehydrogenase and glutathione S-transferase, remained essentially unchanged within the dose range investigated.

Animals↗

PubMed1984

Inhibitory effect of nafenopin upon the development of diethylnitrosamine-induced enzyme-altered foci within the rat liver.

The effect of nafenopin and phenobarbitone upon the distribution of gamma-glutamyltranspeptidase activity and epoxide hydrolase antigenic sites in the liver and upon the development of enzyme-altered foci during hepatocarcinogenesis have been compared. Phenobarbitone induced gamma-glutamyltranspeptidase activity in perilobular hepatocytes. Nafenopin did not alter the distribution of this enzyme. Both compounds appeared to induce epoxide hydrolase; phenobarbitone increased the enzyme content of centrilobular cells, whilst nafenopin altered immunostaining mainly in portal regions. Hepatic lesions were induced by treating one day-old rats with diethylnitrosamine. Phenobarbitone and nafenopin were then administered in the diet upon weaning. Animals were killed after either 2, 4 or 8 weeks feeding and liver sections were stained for the two enzymes. Only sections from nitrosamine-treated animals contained enzyme-altered foci. In general, gamma-glutamyltranspeptidase-containing foci stained also for epoxide hydrolase; but many hydrolase-positive foci did not stain for gamma-glutamyltranspeptidase activity. Phenobarbitone treatment stimulated the formation of enzyme-altered foci. This effect was more marked in male animals. Nafenopin treatment suppressed the development of foci at all time points, such that less hepatic lesions were seen than in animals which received only diethylnitrosamine. The results cast doubt upon the generality of gamma-glutamyltranspeptidase as a marker for preneoplastic lesions within the liver.

Animals↗

PubMed1984

Use of primary cultures of adult rat hepatocytes to investigate mechanisms of action of nafenopin, a hepatocarcinogenic peroxisome proliferator.

The effect of nafenopin upon primary cultures of adult rat hepatocytes has been investigated. Nafenopin treatment resulted in a proliferation of peroxisomes within the cultured cells. This proliferation was the result of an increase in both the number and size of the peroxisomes. Nafenopin treatment also caused an increased level of thymidine incorporation into the cultures, which was a consequence of replicative DNA synthesis rather than DNA repair. Finally, nafenopin appeared to delay the appearance of gamma-glutamyltranspeptidase activity within the cultured cells. Consequently three effects of nafenopin upon the liver were reproduced using monolayers of adult rat hepatocytes, which suggests that this culture system may be useful to further investigate the molecular processes underlying peroxisome proliferation, and their involvement in the hepatocarcinogenicity of peroxisome proliferators.

Animals↗

PubMed1983

Epoxide hydrolase activity in isolated peroxisomes of mouse liver.

Using trans-stilbene oxide as substrate, the subcellular distribution of epoxide hydrolase was investigated in livers from DBA/2 mice. The highest specific activities were found in cytosolic and light mitochondrial fractions. Isopycnic subfractionation of the light mitochondrial fraction showed that the organelle-bound trans-stilbene oxide hydrolase is localized in peroxisomes.

Animals↗

PubMed1983

Functional supersensitivity to adrenergic agonists in the rat after DSP-4, a selective noradrenergic neurotoxin.

Rats treated with DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], a selective noradrenergic neurotoxin, showed no differences compared to control rats in the number of head dips, a measure of exploratory behavior. Since a previous neurochemical investigation had demonstrated that DSP-4 rats have supersensitive alpha 2- and beta-adrenergic receptors in certain regions of the central nervous system, the behavior of these animals was also examined after the injection of clonidine, an alpha 2 agonist, and clenbuterol, a beta agonist. These drugs reduced, in a dose-dependent manner, the head-dipping of both control and DSP-4 rats. However, this effect was of greater magnitude in DSP-4 animals. Control experiments suggested that the response to clonidine and clenbuterol was mediated centrally by alpha 2 and beta receptors, respectively. Other behavioral experiments with agonists of the dopaminergic and serotoninergic systems indicated that these neurotransmitter systems were unchanged in DSP-4 animals. The results are discussed in terms of the selective action of DSP-4 and the responsiveness of DSP-4 rats to adrenergic agonists. The DSP-4-treated rat may constitute a new model of functional supersensitivity to adrenergic agonists.

Amines↗

PubMed1982

Inactivation of a diol epoxide by dihydrodiol dehydrogenase but not by two epoxide hydrolases.

The mutagenicity of r-8,t-9-dihydroxy-t-10, 11-oxy-8,9,10,11-tetrahydrobenz[a]anthracene (BA-8,9-diol 10, 11-oxide) toward Salmonella typhimurium TA 100 is not decreased by the presence of large amounts of highly purified microsomal or cytosolic epoxide hydrolase. However, highly purified dihydrodiol dehydrogenase inactivates this diol epoxide, which is a major DNA-binding metabolite of benz[a]anthracene. The K-region epoxide, benz[a]anthracene 5,6-oxide (BA 5,6-oxide) is efficiently inactivated by microsomal epoxide hydrolase, is much less readily inactivated by cytosolic epoxide hydrolase, and is not inactivated by dihydrodiol dehydrogenase. This inactivation of a diol epoxide by dihydrodiol dehydrogenase points to a new significance of this enzyme and a new level of control for diol epoxides.

Alcohol Oxidoreductases↗

PubMed1982

Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment.

The distribution of rat liver epoxide hydrolase in various subcellular fractions was investigated by immuno-electron-microscopy. Ferritin-linked monospecific anti-(epoxide hydrolase) immunoglobulins bound specifically to the cytoplasmic surfaces of total microsomal preparations and smooth and rough microsomal fractions as well as the nuclear envelope. Specific binding was not observed when the ferritin conjugates were incubated with peroxisomes, lysosomes and mitochondria. The average specific ferritin load of the individual subcellular fractions correlated well with the measured epoxide hydrolase activities. This correlation was observed with fractions prepared from control, phenobarbitone-treated and 2-acetamidofluorene-treated rats.

2-Acetylaminofluorene↗