The specific recognition of DNA modifications by repair endonucleases was used to characterize th... more The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA damage induced by photosensitizers in the presence of visible light. Under cell-free conditions, chemically unrelated photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base loss (sensitive to exonuclease III or endonuclease IV) is low. Therefore the damage is markedly different from that induced by hydroxyl radicals. Mechanistically, the generation of the base modifications sensitive to FPG protein involves singlet oxygen in some, but possibly not all cases, as substituting D2O for H2O increases the reaction yield six-fold in the case of methylene blue, but only 1.4-fold in the case of acridine orange. In plasmids from Salmonella typhimurium strains treated with methylene blue or acridine orange plus light and from Escherichia coli strains treated with acridine orange or proflavin plus light, the same type of damage was observed as under cell-free conditions. In L1210 mouse leukemia cells exposed to acridine orange plus light, the numbers of modifications sensitive to FPG protein and exonuclease III were quantified, in addition to strand breaks, by a modified alkaline elution assay. Again, the number of base modifications sensitive to FPG protein was found to be several-fold higher than the number of strand breaks and sites of base loss. It has to be concluded that the DNA damage in the intact cells is not mediated by hydroxyl radicals or cellular nucleases, but by the same mechanism as operates under cell-free conditions with these agents.
Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acqu... more Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acquisition of the multiple genetic alterations necessary for malignant transformation. In order to study the role of abnormal DNA repair in malignant melanoma, we measured the ability of cell lines from malignant melanoma and that of primary melanocytes to process 4 different kinds of DNA damage (pyrimidine dimers, oxidative DNA lesions, replication errors, and DNA double-strand breaks) using 4 different plasmid assays. Based on the number of chromosomes, the DNA index, and the rates of spontaneous micronuclei, the chromosomal stability in primary melanocytes and the melanoma line LIBR was characterized as being high, intermediate in M1, and low in MeWo. Repair of UVB photoproducts, of oxidative DNA damage, and of replication errors was not impaired in any melanoma line. Using linearized shuttle vector plasmid pZ189, LIBR cells and primary melanocytes exhibited a high efficiency of joining overlapping DNA ends, reflecting proficient repair of DNA double-strand breaks. Joining efficiency was reduced slightly in M1 and 2.9-fold in MeWo. This indicates that in the melanoma cell lines studied here, an increase in chromosomal instability is accompanied by a pronounced impairment in the ability to join DNA ends. Although a small sample was studied, this raises the possibility that defects in DNA end joining may also contribute to genetic instability and chromosome aberrations in melanoma.
European Journal of Inorganic Chemistry, Dec 1, 1979
Eingegangen am 6. November 1978 ,,Dial A" 1 der truns-Erythrinan-Reihe zeigt im Gegensatz zu ,,t>... more Eingegangen am 6. November 1978 ,,Dial A" 1 der truns-Erythrinan-Reihe zeigt im Gegensatz zu ,,t>iol B' 3 der cis-Reihe ein ungewohnliches Verhalten. Durch Acetylierung entsteht unter Umlagerung des Ringsystems das Diacetat 17, dessen Verseifung wieder zum Ausgangsmaterial 1 zuriickfuhrt. Die Stereochemie der umkehrbaren Gerustumlagerung zeigen die Formcln l a und 17a. Die Ringerweiterung zu 17 erkennt man im 'H-NMR-Spektrum an der charakteristischen Hochfeldverschiebung des Signals fur das aromatische Proton an C-14. A Reversible Rearrangement of the trans-Erytbrinane Ring System, I" First Observation and Attempt of an Interpretation The "diol A" (1) with trans-erythrinane structure has an unusual reactivity, compared with "diol B" (3) which belongs to the cis-series. O n acetylation, 1 yields the diacetate 17 due to a rearrangement. Hydrolysis of 17 regenerates the starting material 1. Structures 1 a and 17a illustrate the stereochemistry of the reversible rearrangement. The ring-enlargement of 1 to 17 is seen in the 'H-NMR spectra by a characteristic high-field shift of the signal of the aromatic proton at C-14. Konstitution und Konfiguration der als Diol A und B bezeichneten Dihydroxylactame 1 und 3 der Erythrinan-Reihe Vilhuber 3 , beschrieb die Umsetzung des Bromketolactams 2 mit Kaliumhydroxid in Diethylenglycol bei 150 "C unter Bildung zweier stereoisomerer Dihydroxylactame C18Hz3N05 und eines Hydroxyketolactams 4, das schon Seidel"' erhalten hatte. Die Zugehorigkeit der Verbindungen, die wir als Diol A und B bzw. Ketol A bezeichnet haben, zur cis-oder trans-Erythrinan-Reihe blieb zunachst unsicher '). Diol A ist das Hauptprodukt der Umsetzung und steht irn Mittelpunkt der folgenden Untersuchungen, seine Ausbeute liegt bei chromatographischer Aufarbeitung urn 80% und bei direkter Kristallisation nach Umkristallisieren um 65% 'I. Bei chromatographischer Auftrennung fallen als Nebenprodukte Diol B (9Y0), Ketol A (4%) und das als Ausgangsmaterial fur 2 dienende Ketoenollactam 5 (7%) an 'I, Geht man bei obigem Experiment mit der Temperatur bis auf 80°C zuriick, so sinkt die Ausbeute an Diol A zugunsten von Ketol A, wahrend Diol B verschwindet. Fur die folgenden Untersuchungen hat Diol B der FormeI 3 nur untergeordnete Bedeutung, doch sind seine chemischen und spektroskopischen Eigenschaften von unmittel
The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,... more The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,2-dioxetane (HTMD) and 3-(N-[4-pyridino]carbamoyl)methyl-3,4,4-trimethyl-1,2-dioxetane , (APD), in mammalian cell was studied. Both dioxetanes, which are efficient sources of triplet-excited ketones on thermal decomposition, induced morphological transformation in Syrian hamster embryo (SHE) fibroblasts. Unscheduled DNA synthesis in SHE and in HeLa cells could not be detected with these dioxetanes, but the number of micronuclei scored after the first mitosis was dose-dependently increased. Single-strand breaks but not Micrococcus luteus u.v.-endonuclease sensitive sites were observed by alkaline elution in HL-60 cells when treated with sub-lethal doses of HTMD and APD. A possible mechanism for the transformation mediated by DNA and chromosomal damage as well as the intermediacy of triplet carbonyls in these events are discussed.
Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in ... more Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in the tumorigenesis of UV-induced skin cancer. Different wavelengths of UV radiation cause different kinds of DNA damage and mutations. UVB (290-320 nm) generates pyrimidine dimers by direct excitation of the DNA molecule. UVA (320-400 nm) can damage the DNA only indirectly through a photosensitized reaction. This indirect action is mediated mainly by singlet oxygen, which generates purine base modifications, and has been implicated in the carcinogenic effects of UVA. In order to study the processing of directly and indirectly UV-induced DNA damage in human cells, we first treated the replicating plasmid pRSVcat with up to 10 kJ/m2 UVB or with the photosensitizer methylene blue plus visible light (which generates singlet oxygen) in vitro. Then, the damaged plasmid was transfected into normal or repair deficient xeroderma pigmentosum complementation group A (XP-A) cells. DNA repair was assessed by measuring activity of reactivated chloramphenicol acetyltransferase (CAT) enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 days. While XP-A cells exhibited a significantly reduced repair of UVB-induced DNA damage, they showed a normal repair of singlet oxygen-induced DNA damage. This indicates a differential DNA repair pathway for directly and indirectly UV-induced DNA damage in human cells. Irradiation of the plasmid with UVA alone did not result in a genotoxic effect. Only in conjunction with a cell extract, which provides all candidate cellular photosensitizers, did we find a reduced CAT activity after transfection. This indicates that the genotoxicity of UVA is mediated by a cellular photosensitizer.
The Cockayne syndrome B protein (CSB) has long been known to be involved in the repair of DNA mod... more The Cockayne syndrome B protein (CSB) has long been known to be involved in the repair of DNA modifications that block the RNA polymerase in transcribed DNA sequences (transcription-coupled repair). Recent evidence suggests that it also has a more general role in the repair of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2 0-deoxyguanosine (8-oxoG). In mammalian cells, 8-oxoG is a substrate of the repair glycosylase OGG1. Mice without this enzyme accumulate 8-oxoG in the genome and have elevated spontaneous mutation rates. To elucidate the role of CSB in the prevention of mutations by oxidative DNA base damage, we have generated mice that are deficient in Csb or Ogg1 or both genes and carry a non-transcribed bacterial lacI gene for mutation analysis (Big Blue mice). Our results indicate that the overall spontaneous mutation frequencies in the livers of Csb m/m / Ogg1 À/À-mice are elevated not only compared with heterozygous control mice (factor 3.3), but also with Ogg1 À/À-animals (factor 1.6). Sequence analysis revealed that the additional mutations caused by CSB deficiency in an Ogg1 À/À background are mostly G:C to T:A transversions and small deletions. For all mouse strains, the background levels of oxidative purine modifications in the livers correlate linearly with the numbers of G:C to T:A transversions observed. The data indicate that CSB is involved in the inhibition of mutations caused by spontaneous oxidative DNA base damage in a nontranscribed gene.
ity toward ultraviolet light and bifunctional DNA alkyl-Fanconi anemia (FA) is an autosomal reces... more ity toward ultraviolet light and bifunctional DNA alkyl-Fanconi anemia (FA) is an autosomal recessive dis-ating agents was detected in lymphocytes [7] and in order involving progressive pancytopenia, skeletal cultured fibroblasts [8-11]. malformations, and a predisposition to leukemia. Primary lymphocyte and fibroblast cultures derived The in vitro growth of FA fibroblasts is impaired, due from patients with FA grow rather poorly under stanto a defective G2 phase traverse of the cell cycle. Anadard cell culture conditions [11] and exhibit oxygenlyzing the cell cycle of lymphoid cell lines (LCLs) obdependent chromosomal instability [12]. Cultured fitained from peripheral blood of FA patients by transbroblasts from FA patients exhibit a strong oxygen hyformation with Epstein-Barr virus, we found a simipersensitivity as monitored by serial passaging and cell lar G2 phase defect, which was dependent upon the cloning experiments [13]. By flow cytometric analysis oxygen concentration. In addition, FA cells exhibited an elevated level of G2 phase cells was found in FA hypersensitivity toward cis-dichlorodiammineplatifibroblast cultures [14]. The proliferation rate of FA num and mitomycin C, and moderate sensitivity tofibroblasts is too low to allow a full analysis of the cell ward trans-dichlorodiammineplatinum. FA cells, cycle kinetic mechanism underlying this growth deficit. however, showed no elevated sensitivity toward Peripheral blood lymphocytes, on the other hand, were paraquat, an intracellular generator of superoxide successfully analyzed by continuous bromodeoxyuriradicals, or cumene hydroperoxide, a model organic dine labeling and subsequent bivariate Hoechst 33258/ peroxide. Chelating iron with low concentrations of ethidium bromide flow cytometry [14]. This very inforo-phenanthrolin improved cell proliferation and G2 mative method revealed a normal mitogen response phase transit of FA cells at 20% oxygen, but little at and a strongly elevated arrest of cells in the S and G2 5% oxygen. LCL cultures from healthy subjects were phase of the cell cycle together with a prolongation of inhibited in their proliferation rate at all concentrathe first cycle G2 phase and a shortening of the followtions of o-phenanthrolin. Exposure to excess iron, on ing G1 phase as the basic cell cycle kinetic aberration the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA in FA lymphocytes [14]. Of the many agents tested, mutation leads to a cell cycle defect, which is ex-only an elevated oxygen concentration in the gas phase pressed in cultures of lymphoid cells from FA paof the cell culture system allowed the mimicking of this tients, and involves hypersensitivity toward bifuncpattern of cell cycle disturbance in fibroblast cultures tional alkylating agents, oxygen, and iron. ᭧ 1996 from healthy donors [15]. Thus, the FA mutation re-Academic Press, Inc. veals itself in primary cell cultures as a defect in cell cycle traverse concomitant with an increased sensitivity toward oxygen.
Basal steady-state levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2&amp... more Basal steady-state levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) are observed in all types of cells, most probably due to a continuous generation of reactive oxygen species (ROS) in the cellular oxygen metabolism, and it has long been suspected that they might play an important role in the initiation of carcinogenesis. Experimental evidence for this assumption can be obtained by studying the effects of a modulation of the steady-state levels, either by in- or decreasing the generation of oxidative DNA damage, on spontaneous mutation rates and cancer incidence. However, clear answers have not yet been obtained by these strategies. It is still doubtful whether an efficient reduction of the in vivo steady-state levels can be achieved by application of antioxidants, and effects observed under oxidative stress conditions (i.e. increased oxidative DNA damage) are inconclusive due to the pronounced epigenetic effects of ROS on signal transduction and gene expression (tumor promotion). In addition, the reliable quantification of the basal levels of oxidative DNA modifications is still a major problem. Recently, the generation of mice deficient in the repair 8-oxoG (ogg1-/- mice) has opened the door for an alternative approach. Results obtained so far indicate that an increase by less than five 8-oxoG residues per 106 bp in the liver of the knockout animals is associated with a two- to threefold higher spontaneous mutation frequency in transgenic genes. However, the increase in the ogg1-/- mice of the steady-state level of 8-oxoG and the spontaneous mutation frequency was only observed in the liver and apparently too small to enhance the spontaneous cancer incidence significantly. The limited effect seems to be due to a back-up repair system for 8-oxoG in the ogg1-/- mice, and it can be expected that the inactivation of this pathway in double-knockout mice will lead to higher effects and a better assessment of the risk associated with endogenous oxidative DNA damage.
The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fi... more The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the nontranscribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of transcription by 8-oxo-G fully depends on 8-oxoguanine DNA glycosylase (OGG1) and, at the same time, does not require the localization of the lesion in the transcribed DNA strand. We propose that the interruption of transcription is induced by base excision repair intermediates and, therefore, could be a common consequence of various DNA base modifications. Concordantly, the non-blocking DNA modification uracil was also found to inhibit transcription, but in an OGG1-independent manner.
Two in-vitro tests - the photo micronucleus test (PMNT) and the photo Comet assay (PCA) - were ev... more Two in-vitro tests - the photo micronucleus test (PMNT) and the photo Comet assay (PCA) - were evaluated to proof their ability to identify the photogenotoxicological potential of chemicals. Thirteen photoactive substances were tested under blind conditions in three to five laboratories in the years 2002 to 2004 in a ring trial coordinated by the Federal Institute for Drugs and Medical Devices (BfArM), Bonn in two independent runs.Statistical methods used in this validation study will be discussed:(1) The definition of useful toxicological endpoints and the handling of the data as a first and very important step in the process of the statistical analysis.(2) The repeatability and reproducibility must be given for the test to be of practical use and has to be assessed.For the interpretation of the results, "individual" prediction models were developed by all of the participating laboratories.(3) Comparison of the experimental findings with the classification of photogenotoxicity shows the sensitivity and specificity of the tests. A 2x2-contingency table proved to be most useful for this purpose.(4) Despite the small number of laboratories, the limited number of replicates, and chemicals used, an attempt is made to develop a general prediction model.
Journal of Cancer Research and Clinical Oncology, 1995
Transforming growth factor-B1 (TGFBI) is a potent growth inhibitor and inducer of apoptotic cell ... more Transforming growth factor-B1 (TGFBI) is a potent growth inhibitor and inducer of apoptotic cell death in hepatocyte cultures and in regressing rat liver. Therefore, TGFBI might provide a powerful new agent for therapy of liver cancer. First we determined endogeneous hepatic TGFBI levels through acid ethanol extraction and quantification in the CC164-bioassay. The concentration of the TGFBI polypeptide in female Wistar rats appeared constant during cyproterone acetate-induced hyperplastic growth of the liver and during the subsequent regression of liver hyperplasia by apoptosis. In hepatocellular carcinoma (obtained after a 17 months treatment of male Wistar rats with the peroxisome proliferator nafenopin, 100mg/kg b.w./day) TGFBI levels were not different from that in the surrounding tissue. This suggests that (pre)neoplastic liver cells still might respond to large doses of exogeneously supplied TGFBI. Then we tested the possible therapeutic benefit of TGFBI. Hepatocarcinogenesis was initiated by a single dose of 7,12-dimethylbenz(a)anthracene (130mg/kg b.w.) in female Wistar rats. After recovery for few weeks rats received eight i.v. applications of TGFB1 (40~g /kg b.w./day) which induced apoptosis more in preneoplastic than in unaltered cells. AS a consequence, number and size of preneoplastic liver foci decreased considerably. Coadministration of the anti-estrogen tamoxifen (8mg/kg b.w./day) exerted an overadditive effect on the growth inhibiting and apoptosis stimulating activities of TGFBI in liver preneoplasia. In conclusion, TGFBI in combination with tamoxifen eliminates preneoplastic hepatocytes through apoptosis and may provide great benefit for cancer therapy.
Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by ... more Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by peroxynitrite. Repair endonucleases allow a convenient quantification of various types of oxidative modifications induced by peroxynitrite, both in cultured cells and in cell-free DNA. The high sensitivity of the assays allows highly ectotoxic exposure conditions to be avoided—as well as the generation of secondary DNA modifications—that often become a problem at high levels of damage because primary DNA oxidation products can be orders of magnitude more reactive than the original bases, as demonstrated for the reaction of 8-hydroxyguanine with singlet oxygen. The ratio of the various types of modification (damage profile) is a fingerprint of the reactive species directly responsible for the DNA damage. Thus, the damage profiles induced by hydroxyl radicals and peroxynitrite are clearly distinguishable from each other, but independent of the reactions used to generate the two species. The fingerprint character of the two damage profiles is further supported by the observation that scavengers and antioxidants influence the various types of modification similarly—that is, do not change the ratios.
Fortschritte der Chemie Organischer Naturstoffe, 1983
The Cneoraceae, a plant family of considerable interest to the natural products chemist, comprise... more The Cneoraceae, a plant family of considerable interest to the natural products chemist, comprises two genera with a total of only three species, two of which we have studied in considerable detail. These two species are Neochamaelea pulverulenta (Vent.) Erdtman (= Cneorum pulverulentum Vent.), a xerophytic shrub native to the Canary Islands, with silver-haired leaves, yellow blossoms and hard stone fruits (Fig. 1), and Cneorum tricoccon L., a shrub native to coastal areas of the western Mediterranean, with hairless leaves, yellow blossoms and red fruits (Fig. 2). We had no means of access to the third species, Cneorum trimerum (Urban) Chodat, which belongs to the flora of Cuba. Taxonomically, the family of Cneoraceae is assigned by A. Takhtajan (1) to the Rutales, and by A. Cronquist (2) to the Sapindales; the botanically interested reader is referred to recent articles by H. Straka et al. (3), and by D. Lobreau-Callen et al. (4).
Wir berichteten kiirzlich iiber neue Bitterstoffe aus Neocharnaelea pulverulenta (Vent.) Erdtm. =... more Wir berichteten kiirzlich iiber neue Bitterstoffe aus Neocharnaelea pulverulenta (Vent.) Erdtm. = Cneorum pulverulentum (Vent.
The specific recognition of DNA modifications by repair endonucleases was used to characterize th... more The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA damage induced by photosensitizers in the presence of visible light. Under cell-free conditions, chemically unrelated photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base loss (sensitive to exonuclease III or endonuclease IV) is low. Therefore the damage is markedly different from that induced by hydroxyl radicals. Mechanistically, the generation of the base modifications sensitive to FPG protein involves singlet oxygen in some, but possibly not all cases, as substituting D2O for H2O increases the reaction yield six-fold in the case of methylene blue, but only 1.4-fold in the case of acridine orange. In plasmids from Salmonella typhimurium strains treated with methylene blue or acridine orange plus light and from Escherichia coli strains treated with acridine orange or proflavin plus light, the same type of damage was observed as under cell-free conditions. In L1210 mouse leukemia cells exposed to acridine orange plus light, the numbers of modifications sensitive to FPG protein and exonuclease III were quantified, in addition to strand breaks, by a modified alkaline elution assay. Again, the number of base modifications sensitive to FPG protein was found to be several-fold higher than the number of strand breaks and sites of base loss. It has to be concluded that the DNA damage in the intact cells is not mediated by hydroxyl radicals or cellular nucleases, but by the same mechanism as operates under cell-free conditions with these agents.
Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acqu... more Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acquisition of the multiple genetic alterations necessary for malignant transformation. In order to study the role of abnormal DNA repair in malignant melanoma, we measured the ability of cell lines from malignant melanoma and that of primary melanocytes to process 4 different kinds of DNA damage (pyrimidine dimers, oxidative DNA lesions, replication errors, and DNA double-strand breaks) using 4 different plasmid assays. Based on the number of chromosomes, the DNA index, and the rates of spontaneous micronuclei, the chromosomal stability in primary melanocytes and the melanoma line LIBR was characterized as being high, intermediate in M1, and low in MeWo. Repair of UVB photoproducts, of oxidative DNA damage, and of replication errors was not impaired in any melanoma line. Using linearized shuttle vector plasmid pZ189, LIBR cells and primary melanocytes exhibited a high efficiency of joining overlapping DNA ends, reflecting proficient repair of DNA double-strand breaks. Joining efficiency was reduced slightly in M1 and 2.9-fold in MeWo. This indicates that in the melanoma cell lines studied here, an increase in chromosomal instability is accompanied by a pronounced impairment in the ability to join DNA ends. Although a small sample was studied, this raises the possibility that defects in DNA end joining may also contribute to genetic instability and chromosome aberrations in melanoma.
European Journal of Inorganic Chemistry, Dec 1, 1979
Eingegangen am 6. November 1978 ,,Dial A" 1 der truns-Erythrinan-Reihe zeigt im Gegensatz zu ,,t>... more Eingegangen am 6. November 1978 ,,Dial A" 1 der truns-Erythrinan-Reihe zeigt im Gegensatz zu ,,t>iol B' 3 der cis-Reihe ein ungewohnliches Verhalten. Durch Acetylierung entsteht unter Umlagerung des Ringsystems das Diacetat 17, dessen Verseifung wieder zum Ausgangsmaterial 1 zuriickfuhrt. Die Stereochemie der umkehrbaren Gerustumlagerung zeigen die Formcln l a und 17a. Die Ringerweiterung zu 17 erkennt man im 'H-NMR-Spektrum an der charakteristischen Hochfeldverschiebung des Signals fur das aromatische Proton an C-14. A Reversible Rearrangement of the trans-Erytbrinane Ring System, I" First Observation and Attempt of an Interpretation The "diol A" (1) with trans-erythrinane structure has an unusual reactivity, compared with "diol B" (3) which belongs to the cis-series. O n acetylation, 1 yields the diacetate 17 due to a rearrangement. Hydrolysis of 17 regenerates the starting material 1. Structures 1 a and 17a illustrate the stereochemistry of the reversible rearrangement. The ring-enlargement of 1 to 17 is seen in the 'H-NMR spectra by a characteristic high-field shift of the signal of the aromatic proton at C-14. Konstitution und Konfiguration der als Diol A und B bezeichneten Dihydroxylactame 1 und 3 der Erythrinan-Reihe Vilhuber 3 , beschrieb die Umsetzung des Bromketolactams 2 mit Kaliumhydroxid in Diethylenglycol bei 150 "C unter Bildung zweier stereoisomerer Dihydroxylactame C18Hz3N05 und eines Hydroxyketolactams 4, das schon Seidel"' erhalten hatte. Die Zugehorigkeit der Verbindungen, die wir als Diol A und B bzw. Ketol A bezeichnet haben, zur cis-oder trans-Erythrinan-Reihe blieb zunachst unsicher '). Diol A ist das Hauptprodukt der Umsetzung und steht irn Mittelpunkt der folgenden Untersuchungen, seine Ausbeute liegt bei chromatographischer Aufarbeitung urn 80% und bei direkter Kristallisation nach Umkristallisieren um 65% 'I. Bei chromatographischer Auftrennung fallen als Nebenprodukte Diol B (9Y0), Ketol A (4%) und das als Ausgangsmaterial fur 2 dienende Ketoenollactam 5 (7%) an 'I, Geht man bei obigem Experiment mit der Temperatur bis auf 80°C zuriick, so sinkt die Ausbeute an Diol A zugunsten von Ketol A, wahrend Diol B verschwindet. Fur die folgenden Untersuchungen hat Diol B der FormeI 3 nur untergeordnete Bedeutung, doch sind seine chemischen und spektroskopischen Eigenschaften von unmittel
The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,... more The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,2-dioxetane (HTMD) and 3-(N-[4-pyridino]carbamoyl)methyl-3,4,4-trimethyl-1,2-dioxetane , (APD), in mammalian cell was studied. Both dioxetanes, which are efficient sources of triplet-excited ketones on thermal decomposition, induced morphological transformation in Syrian hamster embryo (SHE) fibroblasts. Unscheduled DNA synthesis in SHE and in HeLa cells could not be detected with these dioxetanes, but the number of micronuclei scored after the first mitosis was dose-dependently increased. Single-strand breaks but not Micrococcus luteus u.v.-endonuclease sensitive sites were observed by alkaline elution in HL-60 cells when treated with sub-lethal doses of HTMD and APD. A possible mechanism for the transformation mediated by DNA and chromosomal damage as well as the intermediacy of triplet carbonyls in these events are discussed.
Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in ... more Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in the tumorigenesis of UV-induced skin cancer. Different wavelengths of UV radiation cause different kinds of DNA damage and mutations. UVB (290-320 nm) generates pyrimidine dimers by direct excitation of the DNA molecule. UVA (320-400 nm) can damage the DNA only indirectly through a photosensitized reaction. This indirect action is mediated mainly by singlet oxygen, which generates purine base modifications, and has been implicated in the carcinogenic effects of UVA. In order to study the processing of directly and indirectly UV-induced DNA damage in human cells, we first treated the replicating plasmid pRSVcat with up to 10 kJ/m2 UVB or with the photosensitizer methylene blue plus visible light (which generates singlet oxygen) in vitro. Then, the damaged plasmid was transfected into normal or repair deficient xeroderma pigmentosum complementation group A (XP-A) cells. DNA repair was assessed by measuring activity of reactivated chloramphenicol acetyltransferase (CAT) enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 days. While XP-A cells exhibited a significantly reduced repair of UVB-induced DNA damage, they showed a normal repair of singlet oxygen-induced DNA damage. This indicates a differential DNA repair pathway for directly and indirectly UV-induced DNA damage in human cells. Irradiation of the plasmid with UVA alone did not result in a genotoxic effect. Only in conjunction with a cell extract, which provides all candidate cellular photosensitizers, did we find a reduced CAT activity after transfection. This indicates that the genotoxicity of UVA is mediated by a cellular photosensitizer.
The Cockayne syndrome B protein (CSB) has long been known to be involved in the repair of DNA mod... more The Cockayne syndrome B protein (CSB) has long been known to be involved in the repair of DNA modifications that block the RNA polymerase in transcribed DNA sequences (transcription-coupled repair). Recent evidence suggests that it also has a more general role in the repair of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2 0-deoxyguanosine (8-oxoG). In mammalian cells, 8-oxoG is a substrate of the repair glycosylase OGG1. Mice without this enzyme accumulate 8-oxoG in the genome and have elevated spontaneous mutation rates. To elucidate the role of CSB in the prevention of mutations by oxidative DNA base damage, we have generated mice that are deficient in Csb or Ogg1 or both genes and carry a non-transcribed bacterial lacI gene for mutation analysis (Big Blue mice). Our results indicate that the overall spontaneous mutation frequencies in the livers of Csb m/m / Ogg1 À/À-mice are elevated not only compared with heterozygous control mice (factor 3.3), but also with Ogg1 À/À-animals (factor 1.6). Sequence analysis revealed that the additional mutations caused by CSB deficiency in an Ogg1 À/À background are mostly G:C to T:A transversions and small deletions. For all mouse strains, the background levels of oxidative purine modifications in the livers correlate linearly with the numbers of G:C to T:A transversions observed. The data indicate that CSB is involved in the inhibition of mutations caused by spontaneous oxidative DNA base damage in a nontranscribed gene.
ity toward ultraviolet light and bifunctional DNA alkyl-Fanconi anemia (FA) is an autosomal reces... more ity toward ultraviolet light and bifunctional DNA alkyl-Fanconi anemia (FA) is an autosomal recessive dis-ating agents was detected in lymphocytes [7] and in order involving progressive pancytopenia, skeletal cultured fibroblasts [8-11]. malformations, and a predisposition to leukemia. Primary lymphocyte and fibroblast cultures derived The in vitro growth of FA fibroblasts is impaired, due from patients with FA grow rather poorly under stanto a defective G2 phase traverse of the cell cycle. Anadard cell culture conditions [11] and exhibit oxygenlyzing the cell cycle of lymphoid cell lines (LCLs) obdependent chromosomal instability [12]. Cultured fitained from peripheral blood of FA patients by transbroblasts from FA patients exhibit a strong oxygen hyformation with Epstein-Barr virus, we found a simipersensitivity as monitored by serial passaging and cell lar G2 phase defect, which was dependent upon the cloning experiments [13]. By flow cytometric analysis oxygen concentration. In addition, FA cells exhibited an elevated level of G2 phase cells was found in FA hypersensitivity toward cis-dichlorodiammineplatifibroblast cultures [14]. The proliferation rate of FA num and mitomycin C, and moderate sensitivity tofibroblasts is too low to allow a full analysis of the cell ward trans-dichlorodiammineplatinum. FA cells, cycle kinetic mechanism underlying this growth deficit. however, showed no elevated sensitivity toward Peripheral blood lymphocytes, on the other hand, were paraquat, an intracellular generator of superoxide successfully analyzed by continuous bromodeoxyuriradicals, or cumene hydroperoxide, a model organic dine labeling and subsequent bivariate Hoechst 33258/ peroxide. Chelating iron with low concentrations of ethidium bromide flow cytometry [14]. This very inforo-phenanthrolin improved cell proliferation and G2 mative method revealed a normal mitogen response phase transit of FA cells at 20% oxygen, but little at and a strongly elevated arrest of cells in the S and G2 5% oxygen. LCL cultures from healthy subjects were phase of the cell cycle together with a prolongation of inhibited in their proliferation rate at all concentrathe first cycle G2 phase and a shortening of the followtions of o-phenanthrolin. Exposure to excess iron, on ing G1 phase as the basic cell cycle kinetic aberration the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA in FA lymphocytes [14]. Of the many agents tested, mutation leads to a cell cycle defect, which is ex-only an elevated oxygen concentration in the gas phase pressed in cultures of lymphoid cells from FA paof the cell culture system allowed the mimicking of this tients, and involves hypersensitivity toward bifuncpattern of cell cycle disturbance in fibroblast cultures tional alkylating agents, oxygen, and iron. ᭧ 1996 from healthy donors [15]. Thus, the FA mutation re-Academic Press, Inc. veals itself in primary cell cultures as a defect in cell cycle traverse concomitant with an increased sensitivity toward oxygen.
Basal steady-state levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2&amp... more Basal steady-state levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) are observed in all types of cells, most probably due to a continuous generation of reactive oxygen species (ROS) in the cellular oxygen metabolism, and it has long been suspected that they might play an important role in the initiation of carcinogenesis. Experimental evidence for this assumption can be obtained by studying the effects of a modulation of the steady-state levels, either by in- or decreasing the generation of oxidative DNA damage, on spontaneous mutation rates and cancer incidence. However, clear answers have not yet been obtained by these strategies. It is still doubtful whether an efficient reduction of the in vivo steady-state levels can be achieved by application of antioxidants, and effects observed under oxidative stress conditions (i.e. increased oxidative DNA damage) are inconclusive due to the pronounced epigenetic effects of ROS on signal transduction and gene expression (tumor promotion). In addition, the reliable quantification of the basal levels of oxidative DNA modifications is still a major problem. Recently, the generation of mice deficient in the repair 8-oxoG (ogg1-/- mice) has opened the door for an alternative approach. Results obtained so far indicate that an increase by less than five 8-oxoG residues per 106 bp in the liver of the knockout animals is associated with a two- to threefold higher spontaneous mutation frequency in transgenic genes. However, the increase in the ogg1-/- mice of the steady-state level of 8-oxoG and the spontaneous mutation frequency was only observed in the liver and apparently too small to enhance the spontaneous cancer incidence significantly. The limited effect seems to be due to a back-up repair system for 8-oxoG in the ogg1-/- mice, and it can be expected that the inactivation of this pathway in double-knockout mice will lead to higher effects and a better assessment of the risk associated with endogenous oxidative DNA damage.
The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fi... more The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the nontranscribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of transcription by 8-oxo-G fully depends on 8-oxoguanine DNA glycosylase (OGG1) and, at the same time, does not require the localization of the lesion in the transcribed DNA strand. We propose that the interruption of transcription is induced by base excision repair intermediates and, therefore, could be a common consequence of various DNA base modifications. Concordantly, the non-blocking DNA modification uracil was also found to inhibit transcription, but in an OGG1-independent manner.
Two in-vitro tests - the photo micronucleus test (PMNT) and the photo Comet assay (PCA) - were ev... more Two in-vitro tests - the photo micronucleus test (PMNT) and the photo Comet assay (PCA) - were evaluated to proof their ability to identify the photogenotoxicological potential of chemicals. Thirteen photoactive substances were tested under blind conditions in three to five laboratories in the years 2002 to 2004 in a ring trial coordinated by the Federal Institute for Drugs and Medical Devices (BfArM), Bonn in two independent runs.Statistical methods used in this validation study will be discussed:(1) The definition of useful toxicological endpoints and the handling of the data as a first and very important step in the process of the statistical analysis.(2) The repeatability and reproducibility must be given for the test to be of practical use and has to be assessed.For the interpretation of the results, "individual" prediction models were developed by all of the participating laboratories.(3) Comparison of the experimental findings with the classification of photogenotoxicity shows the sensitivity and specificity of the tests. A 2x2-contingency table proved to be most useful for this purpose.(4) Despite the small number of laboratories, the limited number of replicates, and chemicals used, an attempt is made to develop a general prediction model.
Journal of Cancer Research and Clinical Oncology, 1995
Transforming growth factor-B1 (TGFBI) is a potent growth inhibitor and inducer of apoptotic cell ... more Transforming growth factor-B1 (TGFBI) is a potent growth inhibitor and inducer of apoptotic cell death in hepatocyte cultures and in regressing rat liver. Therefore, TGFBI might provide a powerful new agent for therapy of liver cancer. First we determined endogeneous hepatic TGFBI levels through acid ethanol extraction and quantification in the CC164-bioassay. The concentration of the TGFBI polypeptide in female Wistar rats appeared constant during cyproterone acetate-induced hyperplastic growth of the liver and during the subsequent regression of liver hyperplasia by apoptosis. In hepatocellular carcinoma (obtained after a 17 months treatment of male Wistar rats with the peroxisome proliferator nafenopin, 100mg/kg b.w./day) TGFBI levels were not different from that in the surrounding tissue. This suggests that (pre)neoplastic liver cells still might respond to large doses of exogeneously supplied TGFBI. Then we tested the possible therapeutic benefit of TGFBI. Hepatocarcinogenesis was initiated by a single dose of 7,12-dimethylbenz(a)anthracene (130mg/kg b.w.) in female Wistar rats. After recovery for few weeks rats received eight i.v. applications of TGFB1 (40~g /kg b.w./day) which induced apoptosis more in preneoplastic than in unaltered cells. AS a consequence, number and size of preneoplastic liver foci decreased considerably. Coadministration of the anti-estrogen tamoxifen (8mg/kg b.w./day) exerted an overadditive effect on the growth inhibiting and apoptosis stimulating activities of TGFBI in liver preneoplasia. In conclusion, TGFBI in combination with tamoxifen eliminates preneoplastic hepatocytes through apoptosis and may provide great benefit for cancer therapy.
Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by ... more Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by peroxynitrite. Repair endonucleases allow a convenient quantification of various types of oxidative modifications induced by peroxynitrite, both in cultured cells and in cell-free DNA. The high sensitivity of the assays allows highly ectotoxic exposure conditions to be avoided—as well as the generation of secondary DNA modifications—that often become a problem at high levels of damage because primary DNA oxidation products can be orders of magnitude more reactive than the original bases, as demonstrated for the reaction of 8-hydroxyguanine with singlet oxygen. The ratio of the various types of modification (damage profile) is a fingerprint of the reactive species directly responsible for the DNA damage. Thus, the damage profiles induced by hydroxyl radicals and peroxynitrite are clearly distinguishable from each other, but independent of the reactions used to generate the two species. The fingerprint character of the two damage profiles is further supported by the observation that scavengers and antioxidants influence the various types of modification similarly—that is, do not change the ratios.
Fortschritte der Chemie Organischer Naturstoffe, 1983
The Cneoraceae, a plant family of considerable interest to the natural products chemist, comprise... more The Cneoraceae, a plant family of considerable interest to the natural products chemist, comprises two genera with a total of only three species, two of which we have studied in considerable detail. These two species are Neochamaelea pulverulenta (Vent.) Erdtman (= Cneorum pulverulentum Vent.), a xerophytic shrub native to the Canary Islands, with silver-haired leaves, yellow blossoms and hard stone fruits (Fig. 1), and Cneorum tricoccon L., a shrub native to coastal areas of the western Mediterranean, with hairless leaves, yellow blossoms and red fruits (Fig. 2). We had no means of access to the third species, Cneorum trimerum (Urban) Chodat, which belongs to the flora of Cuba. Taxonomically, the family of Cneoraceae is assigned by A. Takhtajan (1) to the Rutales, and by A. Cronquist (2) to the Sapindales; the botanically interested reader is referred to recent articles by H. Straka et al. (3), and by D. Lobreau-Callen et al. (4).
Wir berichteten kiirzlich iiber neue Bitterstoffe aus Neocharnaelea pulverulenta (Vent.) Erdtm. =... more Wir berichteten kiirzlich iiber neue Bitterstoffe aus Neocharnaelea pulverulenta (Vent.) Erdtm. = Cneorum pulverulentum (Vent.
Uploads
Papers by Bernd Epe