[The toxicity of dental amalgam fillings].
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Body Burden↗
Biomedical subjects
F Oesch
Publications and source records attributed to F Oesch.
Enhanced expression of the proto-oncogenes fos and raf in the rhabdomyosarcoma cell line BA-HAN-1C after differentiation induction with retinoic acid and N-methylformamide.
BA-HAN-IC is a clonal rat rhabdomyosarcoma cell line consisting of proliferating mononuclear tumor cells, some of which spontaneously fuse to form terminally differentiated post-mitotic myotubes. Exposure of BA-HAN-IC cells to retinoic acid (RA) or N-methylformamide (NMF) resulted in a significant inhibition of proliferation (p less than 0.001) and in cellular differentiation, as evidenced by a significant increase in the creatine kinase (CK) activity (p less than 0.05) and the number of terminally differentiated post-mitotic myotubes (p less than 0.001). Furthermore, between 5% (NMF) and 30% (RA) of the mononuclear tumor cells exhibited ultrastructural features of rhabdomyogenic differentiation, not observed in their mononuclear counterparts under standard growth conditions. Although BA-HAN-IC cells responded to both inducers of differentiation, differences in time course and magnitude of both increase of differentiation and growth inhibition were observed. These effects of RA and NMF were preceded by a marked enhancement of c-raf expression which became evident 6 and 12 hr after exposure to RA and NMF, respectively, and which persisted throughout the observation period of 5 days. Furthermore, a transient expression of c-fos could be observed 15 and 30 min after exposure to RA. Our results suggest that c-raf expression might be implicated in the differentiation process of BA-HAN-IC tumor cells.
Animals↗
A time course investigation of vitamin A level and lipid composition of the liver endoplasmic reticulum in rats following treatment with congeneric polychlorobiphenyls.
The drug metabolizing enzyme activities, the vitamin A content and the fatty acid composition in the endoplasmic reticulum membrane were studied in rat liver after a single injection of the polychlorobiphenyls (PCBs) 3,3',4,4'-tetrachlorobiphenyl [(3,4)2Cl] or 2,2',4,4',5,5'-hexachloro-biphenyl [(2,4,5)2Cl], 300 mumol/kg each. The microsomal vitamin A level was markedly lowered 3 days after treatment with (3,4)2Cl, a coplanar type inducer of cytochrome P-450. A marked increase in microsomal AHH and UDPGT activities occurred within 3 days after injection of (3,4)2Cl whereas (2,4,5,)2Cl treatment enhanced APDM activity only. Arachidonic, stearic and linoleic acid microsomal contents were enhanced by the two congeners. (3,4)2Cl caused the proportion of docosahexaenoic acid to decrease. No highly significant correlation was found between the vitamin A content and lipid components in the microsomal membrane. However, the vitamin A level was inversely related to the activities of drug metabolizing enzymes induced by coplanar compounds (cytochrome P-450 towards benzo[a]pyrene and UDP glucuronosyl transferase towards 4-nitrophenol).
Animals↗
Phosphorylation of cytochrome P450 isoenzymes in intact hepatocytes and its importance for their function in metabolic processes.
Recent data show that besides the well-known long-term regulation of cytochrome P450-dependent monooxygenase activity by induction there also exists a fast regulation by phosphorylation. This phosphorylation occurs when purified cytochromes P450 are combined with purified protein kinases, and also in intact cells. This process is donor- and acceptor-selective leading to phosphorylation of defined isoenzymes by defined protein kinases. This in turn leads to fast and marked changes in metabolism which are selective for given substrates and regio- and stereo-selective for given positions. This in turn is selectively and differentially influenced by the individual control of the protein kinase in question.
Animals↗
Metabolism of 3-hydroxychrysene by rat liver microsomal preparations.
3-Hydroxychrysene, a metabolite of the polycyclic aromatic hydrocarbon (PAH) chrysene, was metabolised by rat liver microsomal preparations obtained from Arochlor 1254-pretreated rats. Eight major metabolites were isolated by high performance liquid chromatography and characterised by u.v. spectroscopy and a variety of mass spectrometric techniques. The metabolites were unambiguously identified as 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene and 9-hydroxy-r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene and tentatively identified as 3-hydroxy-trans-5,6-dihydroxy-5,6-dihydrochrysene (since chrysene is a symmetrical molecule the 3- and 9-positions are equivalent), 9-hydroxy-trans-3,4-dihydroxy-3,4-dihydrochrysene, 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene, an oxidised phenol and two diphenols. These results indicate that 3-hydroxychrysene can be further metabolised via a number of different pathways including those involving the formation of phenol- and triol-epoxides.
Animals↗
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.
Alcohol Oxidoreductases↗
Modulation of the control of mutagenic metabolites derived from cyclophosphamide and ifosfamide by stimulation of protein kinase A.
The phosphorylation of the 2 major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in intact hepatocytes by the action of the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Under these conditions cyclophosphamide and ifosfamide (which are known to be activated by cytochrome P450 IIB1) were investigated for mutagenicity in Salmonella typhimurium TA1535 and TA100 and for cytotoxicity in TA1535. Cyclophosphamide and ifosfamide were transformed to mutagenic and cytotoxic metabolites by the hepatocytes. The activation of both drugs to mutagens was markedly reduced after pretreatment of the hepatocytes with the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Cyclophosphamide and ifosfamide activation were reduced to 51% and 38% of unstimulated controls respectively, when hepatocytes were incubated for 1 h with N6,O2'-dibutyryl-cAMP in the presence of the phosphodiesterase inhibitor theophylline, and Salmonella typhimurium TA1535 was used. A marked reduction in mutagenicity of cyclophosphamide (35% compared with unstimulated controls) was also observed under different experimental conditions, namely after pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP for 1.5 h without theophylline and using Salmonella typhimurium TA100 as target strain. Continued presence of the cytochrome P450 IIB1 and P450 IIB2 inducer phenobarbital in the stimulation medium increased the mutagenicity of cyclophosphamide and led to an even more marked reduction of mutagenicity by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. In order to investigate whether the observed changes were metabolism-related, the ifosfamide metabolite ifosfamide mustard which does not require metabolic activation by cytochrome P450 was studied under the same conditions. Its mutagenicity was indistinguishable after incubation with N6,O2'-dibutyryl-cAMP-treated or with unstimulated hepatocytes. Also the metabolic formation of cytotoxic metabolites from cyclophosphamide and ifosfamide but not that of ifosfamide mustard was markedly decreased by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. Thus the stimulation of protein kinase A in intact cells has important consequences for the control of genotoxic and cytotoxic metabolites and represents a fast and short-term regulation of it.
Animals↗
Mutagenicity experiments on L-cysteine and D-penicillamine using V79 cells as indicators and for metabolic activation.
Previous studies have shown that cysteine and penicillamine induce gene mutations in Salmonella typhimurium, the effect being strongly potentiated in the presence of mammalian tissue preparations. It has now been demonstrated that homogenate of V79 Chinese hamster cells is an efficient activator of thiol amino acids as well. Nevertheless, L-cysteine and D-penicillamine did not induce gene mutations (acquisition of resistance towards 6-thioguanine) in V79 cells. This was true even in the presence of the most efficient activating system, kidney postmitochondrial fraction. The result suggests the existence of an effective protective system in mammalian cells against the natural amino acid L-cysteine and its therapeutically used derivative, D-penicillamine.
Amino Acids, Sulfur↗
Isolation and characterization of a 60-70-kD plasma membrane glycoprotein involved in the contact-dependent inhibition of growth.
Previous studies have shown that plasma membrane compounds are involved in the contact-dependent inhibition of growth of human diploid fibroblasts. The purification of the active plasma membrane glycoprotein is described in this report. The glycoprotein has an apparent molecular mass of 60-70 kD and, due to differential sialylation, isoelectric points between pH 5.5. and 6.2. Treatment with sialidase yielded one spot in two-dimensional gel electrophoresis with an isoelectric point of 6.3. After removal of the N-glycosidically linked oligosaccharide chains, the apparent molecular mass is reduced by approximately 22 kD. Treatment was diluted NaOH, which removes the O-glycosidically linked portion of oligosaccharides, resulted in a reduction of the apparent molecular mass by approximately 5 kD. The addition of 50 ng/ml of this glycoprotein-for which the term "contactinhibin" is proposed-in immobilized form to sparsely seeded human fibroblasts resulted in a reversible 70-80% inhibition of growth. The inhibition was not confined to human fibroblasts as other cells were also inhibited, with the exclusion of transformed cells, which are refractory to contactinhibin. The inhibitory activity was abolished by treatment with beta-galactosidase or glycopeptidase F, indicating that the glycan moiety is the biologically active part of the molecule. Confluent cultures treated with antibodies raised against contactinhibin were released from the contact-dependent inhibition of growth. In addition to enhanced saturation density, these cultures exhibited a crisscross growth pattern and the formation of foci. Immunocytochemical studies showed that contactinhibin was associated with vimentin. Furthermore, contactinhibin was found to be not expressed in a species- or organ-specific manner.
Animals↗
Comparative tumorigenicity of picene and dibenz[a,h]anthracene in the mouse.
The carcinogenic activity of the two polycyclic aromatic hydrocarbons (PAHs), picene (benzo[a]chrysene) and dibenz[a,h]anthracene (DBA), was determined in NMRI mice by five different experimental protocols in order to find out if picene is a carcinogen as predicted by recent quantum mechanical calculations in contrast to earlier observations which could not confirm any carcinogenic activity of picene. Single s.c. treatment of adult mice with picene or DBA (308 nmol/animal, each) led to the formation of fibrosarcomas in 63.3% of treated animals regardless of the PAH used. Chronic epicutaneous application of both PAHs (total dose 1.36 mumol) to the back of mice resulted in the development of papillomas with a tumor rate of 22% in the case of picene and of 32% in the case of DBA. When newborn mice were s.c. treated once on day 2 of their life with each of the two PAHs (400 nmol/animal), 27.8% of treated animals developed lung adenomas after 40 weeks in the case of picene compared to 92.1% in the case of DBA. Histopathological examination of the tumors in the three experimental models revealed no difference in the type of tumor between picene and DBA. Epicutaneous application of both PAHs (600 nmol/animal) followed by chronic treatment with 12-O-tetradecanoyl-phorbol-13-acetate for 24 weeks led to the formation of papillomas in 93% of animals treated with DBA while picene showed no tumorigenic activity at all. Initiation of tumorigenesis in the two-stage tumor model with 7,12-dimethylbenz[a]anthracene (1 mumol/animal) and chronic treatment with picene (total dose 4.8 mumol) for 24 weeks was equally ineffective in producing tumors in NMRI mice. This rare biological property of picene, which is a complete carcinogen, yet at most a very weak tumor initiator, is explained in terms of its inefficient biotransformation to mutagenic and carcinogenic metabolites as compared to the strong tumor initiator DBA.
Animals↗
A new assay for O6-alkylguanine-DNA-alkyltransferase to determine DNA repair capacities using lambda-phage DNA as substrate.
One O6-methylguanine (O6-meG) was introduced into each BamHI site of lambda-phage DNA as a substrate for the determination of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase. A new assay using as the detection group 32P-labeled phosphate introduced at the 3' position of the modified nucleoside by incorporation of 32P-labeled TTP in the 3'-neighboring position proved highly sensitive: 10(-16) mol of the DNA lesion was still easily detectable. This DNA, which has greater than 1000 bp represents a good model for cellular DNA and was used as a substrate to measure the individual repair capacities for O6-meG in human lymphocytes of 20 healthy male and female donors. There were great inter-individual variations in the activity of this repair protein in man. The influences of age, sex or smoking behavior on the repair capacity of O6-meG were negligible.
Bacteriophage lambda↗
The initiator tRNA acceptance assay as a short-term test for carcinogens. 6. Results with 78 polycyclic aromatic compounds.
A total of 78 polycyclic aromatic compounds (PAH), including pure hydrocarbons, PAH metabolites, aromatic amines and nitroarenes, were tested in the initiator tRNA acceptance assay (tR assay) for carcinogens. Among the pure hydrocarbons, all strong carcinogens were highly active in the tR assay. Some weak carcinogens showed moderate positive responses, others as well as all possible non-carcinogens were inactive. Various PAH metabolites, including phenols, dihydrodiols, arene oxides, dihydrodiol epoxides, quinones and benzylic sulfate esters, were positive as well. Strikingly, however, their effects rarely reached the levels observed with the strong carcinogens among the pure hydrocarbons. Moreover, the correlation with carcinogenicity was less clear, partially due to limitations in the available carcinogenicity data. The activities in the tR assay were also compared with the mutagenicity in Salmonella typhimurium. No appreciable correlation was observed. For example, trans-9,10-dihydroxy-9,10-dihydrobenzo[c]chrysene, in the absence of a mammalian metabolic system, was highly active in the tR assay, but non-mutagenic. Upon addition of rat liver enzymes, the reverse result was obtained. syn-Benzo[c]chrysene-9,10-dihydrodiol-11,12-oxide, on the other hand, was a potent direct mutagen, but required the presence of liver microsomes for a positive response in the tR assay. Thus, the metabolic basis for these two activities is different, and not yet understood for the tR assay. The partial correlations in the tR assay and in the Salmonella mutagenicity assay with carcinogenicity, and the pronounced discrepancies between these in vitro tests, may suggest that they detect different mechanisms involved in carcinogenicity. However, the tR assay was less predictive for the carcinogenicity of PAHs as compared to the previously investigated N-nitroso compounds and mycotoxins.
Carcinogenicity Tests↗
Reduction of glutathione content by 12-O-tetradecanoylphorbol-13-acetate in confluent, but not in sparse cultures of human diploid fibroblasts.
Treatment of confluent cultures of human diploid fibroblasts with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-7) M) resulted in a 70% reduction of the glutathione (GSH) content, compared with untreated controls. The effect, which was dose-dependent, was observed 8 h after the beginning of the treatment could be followed for up to 72 h. On the other hand, GSH reduction was specific for confluent cultures, as the level of glutathione remained unchanged by TPA treatment of sparse cultures. The addition of immobilized plasma membrane proteins to sparsely seeded cells has been shown previously to induce cellular reactions which are characteristic for confluent cultures. It was shown that TPA treatment of sparse cultures grown in the presence of immobilized plasma membrane proteins also resulted in a 70% reduction of glutathione content. These data agree with the postulated involvement of redox reactions in tumor promotion, and point to a central role of cell-cell contacts in the regulation of biochemical events which are critical in tumorigenesis.
Cell Count↗
Modulation of selenium-dependent glutathione peroxidase by perfluorodecanoic acid in rats: effect of dietary selenium.
Forty-eight male Sprague-Dawley rats fed a diet containing 0.4, 0.2 or 1.0 mg of selenium (Se)/kg of diet were injected with a single dose (35 mg/kg) of perfluorodecanoic acid (PFDA) in corn oil and killed 2 wk later. Control animals were pair-fed and treated with an equal volume of vehicle. PFDA treatment significantly increased Se-dependent glutathione peroxidase (Se-GSHPx) activity in liver cytosol of rats fed the 0.04 mg of Se/kg of diet but not in rats fed the other diets. The increase in liver cytosolic Se-GSHPx activity in rats fed 0.04 mg of Se/kg of diet paralleled increases in Se content and serum Se-GSHPx activity. Determination of Se-GSHPx by an enzyme-linked immunosorbent assay showed that PFDA caused a decrease in Se-GSHPx protein in rats fed 0.2 or 1.0 mg of Se/kg of diet but not in rats fed 0.04 mg of Se/kg of diet. Further analysis revealed that the ratio of Se-GSHPx activity to antibody-reactive protein was increased by PFDA in all three groups. The in vitro addition of PFDA directly to the assay mixture for Se-GSHPx activity did not produce any effect. Reduced glutathione was significantly increased by PFDA treatment in all three groups. These data show that PFDA affects the Se content, Se-GSHPx activity and Se-GSHPx protein in rat liver and that the effect is dependent on the dietary/hepatic Se level.
Animals↗
Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds.
Activities of various xenobiotic-metabolizing enzymes were determined in 18 cell lines. Activities of cytochrome P450 reductase, microsomal epoxide hydrolase and glutathione transferase were detectable in all lines. The highest values were similar to the activities found in freshly isolated rat hepatocytes. Catalase activity was also present in all 12 investigated cell lines. Activity of UDP-glucuronosyl transferase was high in some lines, but low or undetectable in others. Activity of cytosolic epoxide hydrolase was not measurable in most lines, and was low in the others. Metabolism of benzo[a]pyrene was observed in eight out of nine examined lines, no activity being found in V79 cells. V79 and three epithelial cell lines were then used as target cells in a genotoxicity assay in which the frequency of micronucleated cells was determined. In V79 cells, 7,12-dimethyl- benz[a]anthracene, benzo[a]pyrene, benzo[a]pyrene-trans-7,8-dihydrodiol, aflatoxin B1, N-nitrosomorpholine and 2-acetylaminofluorene showed negative responses, whereas N-methyl-N'-nitro-N-nitrosoguanidine, 9-hydroxybenzo[a]pyrene, 2-nitrofluorene, dibenz[a,h]anthracene 1,2-catechol, dibenz[a,h]anthracene, 1,2-quinone hydroquinone and p-benzoquinone proved positive in the test. All 13 compounds, however, induced micronuclei in rat intestinal cells (IEC-17 and IEC-18) and in embryonal human liver cells (HuFoe-15). Thus, these epithelial cell lines are capable of activating and detecting a broad spectrum of chemically diverse genotoxic compounds. They may also be useful for the detection of hazardous compounds whose active metabolites are not able to penetrate from the extracellular space into the indicator cell.
Animals↗
Toxicological implications of enzymatic control of reactive metabolites.
Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human requires information of these variables in the system in question and in man. Differences in susceptibility to toxic challenges between species and individuals are often causally linked to differences in these control factors.
Base Sequence↗
Morphological, biochemical, and molecular biological characterization of a rat rhabdomyosarcoma cell line during differentiation induction in vitro.
BA-HAN-1C is a clonal rat rhabdomyosarcoma cell line consisting of proliferating mononuclear tumor cells, some of which spontaneously fuse to form terminally differentiated postmitotic myotubelike giant cells. Exposure to retinoic acid resulted in an inhibition of proliferation and a marked increase in cellular differentiation. The number of myotubelike giant cells significantly increased, and about 30% of the mononuclear tumor cells exhibited morphological features of rhabdomyogenic differentiation which were not observed in the mononuclear cells of untreated cultures. Morphological differentiation was paralleled by an increase in total creatine kinase activity as a biochemical marker of differentiation. These effects of retinoic acid were preceded by an increased expression of proto-oncogene raf and transient expression of proto-oncogene fos. The maximum level of fos expression was observed at 15 min and of raf at 12 hr after exposure to retinoic acid. No expression of the proto-oncogenes src, myb, myc, ros, mos, erbA, and erbB was detected.
Animals↗
Growth control in mammalian cells by cell-cell contacts.
Growth of normal diploid mammalian cells in vitro is strongly regulated by the actual cell density. Cell-cell contacts via specific plasma membrane glycoproteins whose glycan moieties interact with specific receptors has been found to be a main growth regulatory principle. Malignant growth is suggested to result from impaired function of these receptors.
Cell Adhesion↗