Biomedical subjects
F Oesch
Publications and source records attributed to F Oesch.
Modulation of the covalent binding of aryl hydrocarbon metabolites to DNA in vitro after treatment of rats and mice with trans-stilbene oxide.
The effect of trans-stilbene oxide (TSO) induction on the microsome-catalyzed binding of polycyclic aromatic hydrocarbon metabolites to DNA was investigated using two rodent species (Sprague-Dawley rat and C57BL/6N or NMRI Swiss mouse) and 2 different binding substrates (benzo[a]anthracene). It was determined that TSO exerts 2 separate effects on polycyclic aromatic hydrocarbons - it increases the rate of oxidation at the K-region of the molecule due to its induction of specific monooxygenases, and it increases the rate of deactivation of epoxide intermediates by induction of microsomal epoxide hydrolase activity. The importance of these individual effects were determined by inducing monooxygenase activity with BP, altering region specificity and inducing epoxide hydrolase (EH) activity with TSO, assessing the combined inductive effects of TSO and BP, inhibiting EH with 1,1,1-trichloropropene oxide, or increasing its activity by the addition of pure enzyme. This study shows that these effects are similar for both substrates examined, and that the effect of TSO on the binding to DNA of highly carcinogenic bay-region diol-epoxides is multi-faceted, due to its multiple inductive effects.
Ethanol- or acetone-pretreatment of mice strongly enhances the bacterial mutagenicity of dimethylnitrosamine in assays mediated by liver subcellular fraction, but not in host-mediated assays.
The activation of dimethylnitrosamine (DMN) to a bacterial mutagen in liver subcellular fraction and in intrasanguinous host-mediated assays was studied, in particular the effect of pretreatment of the animals with ethanol or acetone. Salmonella typhimurium TA 92 was much more sensitive to DMN mutagenicity than TA 100 and TA 1535 or Escherichia coli WP2uvrA and was used for the main part of the study. Noteworthy, in part already known, features of the in vitro activation are the relatively low pH optimum (pH 6-6.4), the non-linear dose-mutagenic response-relationship and the relatively high doses of DMN required for activation with control preparations. Pretreatment of mice with ethanol or acetone greatly reduced the minimal mutagenically effective concentration of DMN in the in vitro assay. Pretreatment with Aroclor 1254, an inducer frequently used in mutagenicity research, showed little effect when used alone, but reduced the potentiation by acetone. The results of the host-mediated assays substantially differed from those of the in vitro activation assays (a) in the relatively low dose of DMN required for mutagenicity to occur and (b) in the lack of potentiation by acetone-or ethanol-pretreatment. Acetone even led to a marginal decrease in mutagenicity. As a possible explantation for this apparent discrepancy were assume that with the in vitro system the activity of the dilute metabolizing system is limiting for the activation of DMN and induction therefore will increase the mutagenicity, whereas in vivo DMN is quantitatively metabolized in both induced and non-induced animals. The results show that caution has to be taken in the interpretation from in vitro results to the in vivo situation. In particular our in vivo experiments do not support the hypothesis that the induction by ethanol of an activating system with a low Km (which would strongly activate traces of DMN ingested with many foods) is one of the reasons for the increased risk of liver tumors in alcoholics.
Coordinate mutation and transformation of mouse fibroblasts: induction by nitroquinoline oxide and modulation by caffeine.
Mutation and malignant transformation were followed in the same cells. Mouse fibroblasts (C3H 10T 1/2) were mutated and transformed by 4-nitroquinoline-1-oxide with similar, approximately linear dose-responses. The presence of caffeine immediately after exposure to 4-nitroquinoline-1-oxide potently inhibited mutation and transformation at high but not at low doses of 4-nitroquinoline-1-oxide. Whilst the coordinate induction of mutation and transformation could be explained by both a common target (DNA) or a common reactive species hitting several targets, the identical modulation by a DNA repair inhibitor of both end points suggests fundamental similarities in the nature of the lesions leading to mutation and transformation and in the processing of these lesions, implying DNA as target and mutation as one (but not necessarily the sole) required step in transformation.
Large differences in metabolic activation and inactivation of chemically closely related compounds: effects of pure enzymes and enzyme induction on the mutagenicity of the twelve monomethylated benz[a]anthracenes, 7,12-dimethylbenz[a]anthracene and benz[a]anthracene in the Ames test.
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Improvement of the correlation of bacterial mutagenicity with carcinogenicity of benzo(a)pyrene and four of its major metabolites by activation with intact liver cells instead of cell homogenate.
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Rat liver cytoplasmic dihydrodiol dehydrogenase. Purification to apparent homogeneity and properties.
A method is described for the purification of an enzyme, which catalyzes the conversion of benzene dihydrodiol to catechol, from rat liver cytoplasmic fraction to apparent homogeneity. The purification involved (NH4)2SO4 fractionation, DEAE-cellulose chromatography, interfacial salting-in and gel filtration through Sephadex G-100 superfine. The end product, which was purified over 500-fold with a yield of about 14% when compared to rat liver 100,000 X g supernatant, was judged to be homogeneous by several criteria, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical ultracentrifugation, gel filtration, and immunoprecipitation. Physical studies suggested that the protein was a monomer with a molecular weight of 35,000 and one NADPH binding site per molecule. Amino acid analysis showed that the enzyme had a relatively high content of acidic and neutral amino acids in agreement with its isoelectric point which was at pH 6.2 Apparent Km values for benzene dihydrodiol and NADP+ were found to be 2.2 mM and 7.7 microM, respectively. The apparent Vmax value for the conversion of benzene dihydrodiol to catechol using NADP+ as cofactor was calculated to be 6.67 mumol/mg of enzyme/min. Substrate specificity studies showed that, in addition to benzene dihydrodiol, the dehydrogenase could oxidize acenaphthenol and the 3 alpha-hydroxy group of steroids. No activity was observable with a large number of other hydroxylated steroids possessing hydroxy groups at positions 3 beta, 11 beta, 17 alpha, 17 beta, 20 alpha, 20 beta, 21, and 22 of the steroid skeleton. Furthermore, only steroids which contained a 3-keto group and no double bond at the delta 4 position were reduced. This, and the fact that a range of nonsteroidal vicinal diols did not serve as substrates, indicates a relatively narrow substrate specificity. When benzene dihydrodiol was used as substrate, NADP+ was the preferred coenzyme but NAD+ was also accepted, whereas with the hydroxylated steroids the difference between the specific activities with NAD+ and NADP+ or with NADH and NADPH was less striking. The role of the enzyme in the metabolism of carcinogenic polycyclic hydrocarbons is discussed.
Benzo[a]pyrene 4,5-oxide. Discrepancy between induction of sister chromatid exchange and binding to DNA in cultured human fibroblasts.
Benzo[a]pyrene 4,5-oxide was covalently bound to DNA of cultured human fibroblasts and caused sister chromatid exchange. The monooxygenase inhibitor alpha-napthoflavone suppressed this induction of sister chromatic exchange, but did not affect binding to DNA. Control experiments with 4-nitroquinoline 1-oxide showed that alpha-naphthoflavone does not inhibit sister chromatid exchange in general. A more likely explanation for the discrepancy between induction of sister chromatid exchange and binding to DNA is that benzo[a]pyrene 4,5-oxide itself can bind to DNA, but this binding does not lead to a significant increase in sister chromatid exchange. However benzo[a]pyrene 4,5-oxide can be oxidized by monooxygenase to yet unknown products which are potent inducers of sister chromatid exchange. An important conclusion from this is that a biological effect such as the induction of sister chromatid exchange may correlate with the exact nature of DNA binding rather than with total binding, to the point where just measuring total binding may be completely misleading if intended to detect the causes of the biological effect.
Influence of carbamazepine 10,11-oxide on drug metabolizing enzymes.
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Functional heterogeneity of UDP-glucuronyltransferase in rat tissues.
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Influence of foreign compounds on formation and disposition of reactive metabolites.
Many toxic compounds are unreactive and need biotransformation in order to exert their toxic effects. Several enzymes control the formation or disposition of reactive metabolites. Especially well studied is the group of enzymes responsible for the control of reactive epoxides. Such epoxides may bind spontaneously to DNA, RNA and protein. These alterations of critical cellular macromolecules may disturb the normal biochemistry of the cell and lead to cytotoxic, allergenic, mutagenic and carcinogenic effects. Whether these effects will be manifested depends on the chemical reactivity as well as on other properties (geometry, lipophilicity) of the epoxide in question. Enzymes controlling the concentration of epoxides are another important contributing factor. Several microsomal monooxygenases exist. Some monooxygenases preferentially attack large substrates at single sites, specific for each enzyme. Some of these steps produce reactive metabolites; others are detoxification pathways. Enzymes that metabolize the epoxides represent a further determining factor. These enzymes include epoxide hydrolase (EC 3.3.2.3) and glutathione transferases (EC 2.5.1.18), which do not play a purely inactivating role but can, in some cases, act also as coactivating enzymes. Some of these enzymes have been shown to be influenced by foreign compounds. Acute effects by activation and inhibition of the enzymes as well as long-term effects by induction and repression have been observed. Since different foreign compounds differentially influence various enzymes, they can produce changes not only in overall metabolic activity but also changes in metabolite pattern and in selective toxicities.
Species differences in activating and inactivating enzymes related to in vitro mutagenicity mediated by tissue preparations from these species.
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Enzymic hydration of benzene oxide: assay and properties.
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Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules, and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry.
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Factors responsible for the metabolic formation and inactivation of bacterial mutagens from trans-4-acetylaminostilbene.
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Specificity of mouse liver cytosolic epoxide hydrolase for K-region epoxides derived from polycyclic aromatic hydrocarbons.
Mouse liver cytosol epoxide hydrolase, known to be very active for certain alkene oxides, had a specific activity which was 2.1-, 11- and 160-fold lower than that of the microsomal epoxide hydrolase for the arene oxides 7-methylbenz[a]anthracene 5,6-oxide, benz[a]anthracene 5,6-oxide and phenanthrene 9,10-oxide, respectively. For benzo[a]pyrene 4,5-oxide no activity (less than 10 pmol product/mg protein/min) of cytoplasmic epoxide hydrolase was detectable. The specific activity of cytoplasmic epoxide hydrolase was much lower for all K-region epoxides investigated, compared to trans-stilbene oxide used as a positive control and for which a new assay is described. It is concluded from these rates combined with the fact that these lipophilic K-region epoxides are expected to stay preferentially at membranous sites where they are generated, that cytoplasmic epoxide hydrolase plays a minor role for their transformation compared to membrane-bound hydrolase. The data also show that for the substrates investigated the epoxide hydrolase activities in the cytoplasmic and microsomal fractions are complementary to some extent, but there is no quantitative inverse relationship.
Monooxygenase, epoxide hydrolase, and glutathione-S-transferase activities in human lung. Variation between groups of bronchogenic carcinoma and non-cancer patients and interindividual differences.
Activities of microsomal monooxygenases (MO) and epoxide hydrolase (EH) and cytoplasmic glutathione-S-transferases (GST) will contribute to controlling the pool of reactive intermediates, enzymatically derived from polynuclear aromatic hydrocarbons (PAH) within the cells of target organs such as the human lung. Therefore, we studied what interindividual differences exist in these enzyme activities and whether there is a correlation between the activities of these epoxide forming and metabolizing enzymes in preparations from peripheral lung samples and the occurrence of bronchogenic carcinomas in smokers and non-smokers. 57 samples obtained from surgery were studied. Among them were 12 samples from non-smoking patients without cancer as a control group. It is not known whether this control group behaves, with respect to the investigated parameters, identically to fully healthy people, since in all cases indications existed which justified the removal of lung biopsies. Using very sensitive standard assays with benzo[a]pyrene, biphenyl, 7-ethoxyresorufin and 7-ethoxycoumarin as substrates, MO activity could only be determined as O-deethylation of 7-ethoxycoumarin and only after modification of the assay method. Evidence was obtained for the presence of a diffusible, but not dialysible, MO inhibitor in human lung microsomes. The MO activity (substrate: 7-ethoxycoumarin) in this fraction was extremely low in human (100-fold lower than in rat lung preparations), whereas EH (substrate: benzo[a]pyrene 4,5-oxide) was slightly (about 2-fold) higher in human and GST (substrate: 2,4-dinitrochlorobenzene) had similar activities in both species. Interindividual variations of enzyme activities in human lung were considerable: MO, 40-fold: EH, 5-fold; GST 10-fold. Compared to the control group (non-smokers without cancer) MO activities were slightly but significantly higher in lungs from bronchogenic carcinoma patients whether they were smokers (170% of controls, p < 0.0005) or non-smokers (320% of controls p < 0.025). MO activities of smokers without cancer were only very slightly elevated (140%) of controls, p < 0.05). Specific EH activities compared to the control group were slightly but significantly increased in smokers without cancer (160% of controls, p < 0.0125) and in bronchogenic carcinoma patients whether they used tobacco products (130% of controls, p < 0.005) or not (140% of controls, p < 0.05). Specific GST activities showed no significant differences (p > 0.1) between the various groups studied. The substrate specificity of human lung EH, which was studied using five K-region epoxides of various PAH as substrates, corresponded to that in human and rat liver and in human, mouse and rat skin and to the pure enzyme isolated from rat liver. In contrast to rat liver hepatoma preparations, where EH had been shown to be increased in the tumor tissue and had been identified as a preneoplastic antigen, EH activity in lung microsomal preparations from samples of peripheral squamous cell carcinomas of two subjects had in the tumor tissue only one third of the activity of non-diseased areas of the same lung.