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B Epe

Publications and source records attributed to B Epe.

At least 37 records · Page 2Linked to original sources
PubMed1997

Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases.

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.

Chromatography, High Pressure Liquid↗
PubMed1997

Wavelength dependence of oxidative DNA damage induced by UV and visible light.

DNA damage induced by UV radiation and visible light (290-500 nm) in AS52 Chinese hamster cells was analysed by an alkaline elution assay with specific repair endonucleases. Cells were exposed to extensively filtered monochrome or broad-band radiation. Between 290 and 315 nm, the ratio of base modifications sensitive to Fpg protein (i.e. 8-hydroxyguanine and formamidopyrimidines) and T4 endonuclease V (i.e. cyclobutane pyrimidine dimers) was constant (approximately 1:200), indicating that the direct excitation of DNA is responsible for both types of damage in this range of the spectrum. While the yield of pyrimidine dimers per unit dose continued to decrease exponentially beyond 315 nm, the yield of Fpg-sensitive modifications increased to a second maximum between 400 and 450 nm. The damage spectrum in this wavelength range consisted of only a few other modifications (strand breaks, abasic sites and pyrimidine modifications sensitive to endonuclease III) and is attributed to endogenous photosensitizers that give rise to oxidative DNA damage via singlet oxygen and/or type I reactions. The generation of Fpg-sensitive modifications by visible light was not linear with dose but followed a saturation curve. It is calculated that the exposure of the cells to low doses of solar radiation results in the formation of cyclobutane pyrimidine dimers and Fpg-sensitive modifications in a ratio of 10:1.

Animals↗
PubMed1997

Problems in the measurement of 8-oxoguanine in human DNA. Report of a workshop, DNA oxidation, held in Aberdeen, UK, 19-21 January, 1997.

Oxidative DNA damage is widely believed to play a role in cancer aetiology. It is therefore important to be able to assess it, both as an index of cancer risk, and in experiments to test agents with a potential to reduce oxidative damage, such as dietary antioxidants. However, there is an alarming discordance in estimates of concentrations of oxidative damage in human DNA, largely attributable to the kind of method used to measure it. A meeting was held recently at the Rowett Research Institute in Aberdeen to address this problem.

Animals↗
PubMed1997

Photochemical and photobiological studies with acridine and phenanthridine hydroperoxides in cell-free DNA.

The acridine and phenanthridine hydroperoxides 3 and 7 were synthesized as photochemical hydroxyl radical sources for oxidative DNA damage studies. The generation of hydroxyl radicals upon UVA irradiation (lambda = 350 nm) was verified by trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide and benzene. The enzymatic assays of the damage in cell-free DNA from bacteriophage PM2 caused by the acridine and phenanthridine hydroperoxides 3 and 7 under near-UVA irradiation revealed a wide range of DNA modifications. Particularly, extensive single-strand break formation and DNA base modifications sensitive to formamidopyrimidine DNA glycosylase (Fpg protein) were observed. In the photooxidation of calf thymus DNA, up to 0.69 +/- 0.03% 8-oxo-7,8-dihydroguanine was formed by the hydroperoxides 3 and 7 on irradiation, whose yield was reduced up to 40% in the presence of the hydroxyl radical scavengers mannitol and tert-butanol. The acridine and phenanthridine hydroperoxides 3 and 7 also induce DNA damage through the type I photooxidation process, for which photoinduced electron transfer from 2'-deoxyguanosine to the singlet states of 3 and 7 was estimated by the Rehm-Weller equation to possess a negative Gibb's free energy of ca -5 kcal/ mol. Control experiments with the sensitizers acridine 1 and the acridine alcohol 4 in calf thymus and PM2 DNA confirmed the photosensitizing propensity of the UVA-absorbing chromophores. The present study emphasizes that for the development of selective and efficient photochemical hydroxyl radical sources, chromophores with low photosensitizing ability must be chosen to avoid type I and type II photooxidation processes.

Cell-Free System↗
PubMed1997

Photochemical and photobiological studies of a furonaphthopyranone as a benzo-spaced psoralen analog in cell-free and cellular DNA.

Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glycosylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29 +/- 0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D2O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert-butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (Ered = -1.93 +/- 0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (delta GET ca -6 kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures.

Animals↗
PubMed1996

DNA damage by peroxynitrite characterized with DNA repair enzymes.

The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relatively low. This DNA damage profile caused by peroxynitrite is significantly different from that obtained with hydroxyl radicals or with singlet molecular oxygen. The effects of various radical scavengers and other additives (t-butanol, selenomethionine, selenocystine, desferrioxamine) were the same for single-strand breaks and Fpg-sensitive modifications and indicate that a single reactive intermediate but not peroxynitrite itself is responsible for the damage.

Butanols↗
PubMed1996

Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA.

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen. Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by approximately 50%. A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival. No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells. In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark.

Acridines↗
PubMed1996

Cell cycle defect in connection with oxygen and iron sensitivity in Fanconi anemia lymphoblastoid cells.

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. The in vitro growth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein-Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity toward cis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity toward trans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations of o-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations of o-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.

Alkylating Agents↗
PubMed1995

Genotoxicity induced by furocoumarin hydroperoxides in mammalian cells upon UVA irradiation.

The mutagenicity and micronucleus induction by the furocoumarin hydroperoxides 1a and 2a and, for comparison, their corresponding alcohols 1b and 2b were investigated in L5178Y tk+/- mouse lymphoma cells and AS52 Chinese hamster ovary cells. The furocoumarin hydroperoxide 1a enhanced the micronucleus frequency in L5178Y tk+/- mouse lymphoma cells significantly, while its alcohol 1b exhibited a rather moderate effect. In contrast, both the furocoumarin hydroperoxide 2a and its alcohol 2b induced high frequencies of micronuclei. Only the furocoumarin hydroperoxide 1a, but not its alcohol 1b, was mutagenic in both L5178Y tk+/- and AS52 cells. On the other hand, the furocoumarin hydroperoxide 2a and its alcohol 2b were mutagenic. For all furocoumarin derivatives 1a,b and 2a,b no mutagenicity and genotoxicity was observed in the absence of UVA light. The multifunctional furocoumarin hydroperoxides may serve as potential photochemotherapeutic agents.

Animals↗
PubMed1995

Transcription factor NF-kappa B is activated by photosensitization generating oxidative DNA damages.

Reactive oxygen intermediates like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and, then, in the activation and replication of human immunodeficiency virus (HIV)-1 in human cells. Because H2O2 can be converted into the highly reactive OH. at various locations inside the cells, we started to investigate the generation of Reactive oxygen intermediates by photosensitization. This technique is based on the use of a photosensitizer which is a molecule absorbing visible light and which can be located at various sites inside the cell depending on its physicochemical properties. In this work, we used proflavine (PF), a cationic molecule having a high affinity for DNA, capable of intercalating between DNA base pairs. Upon visible light irradiation, intercalated PF molecules oxidize guanine residues and generate DNA single-strand breaks. In lymphocytes or monocytes latently infected with HIV-1 (ACH-2 or U1, respectively), this photosensitizing treatment induced a cytotoxicity, an induction of NF-kappa B, and a reactivation of HIV-1 in cells surviving the treatment. NF-kappa B induction by PF-mediated photosensitization was not affected by the presence of N-acetyl-L-cysteine while strong inhibition was recorded when the induction was triggered by H2O2 or by phorbol 12-myristate 13-acetate. Another transcription factor like AP-1 is less activated by this photosensitizing treatment. In comparison with other inducing treatments, such as phorbol 12-myristate 13-acetate or tumor necrosis factor alpha, the activation of NF-kappa B is slow, being optimal 120 min after treatment. These kinetic data were obtained by following, on the same samples, both the appearance of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in cytoplasmic extracts. These data allow us to postulate that signaling events, initiated by DNA oxidative damages, are transmitted into the cytoplasm where the inactive NF-kappa B factor is resident and allow the translocation of p50/p65 subunits of NF-kappa B to the nucleus leading to HIV-1 gene expression.

Acetylcysteine↗
PubMed1995

Processing of directly and indirectly ultraviolet-induced DNA damage in human cells.

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.

Cell Line, Transformed↗
PubMed1995

Oxidative DNA damage induced by potassium bromate under cell-free conditions and in mammalian cells.

The oxidative DNA damage induced by the renal carcinogen potassium bromate (KBrO3) in cultured mammalian cells and in a cell-free system was characterized by means of various repair endonucleases. Under cell-free conditions, no modifications were induced by KBrO3 alone, but extensive DNA damage was observed in the presence of glutathione (GSH). The DNA damage was found to consist mostly of base modifications sensitive to Fpg protein (formamidopyrimidine-DNA glycosylase). HPLC analysis demonstrated that many of the modifications were 7,8-dihydro-8-oxoguanine(8-hydroxyguanine) residues. Single-strand breaks, sites of base loss (AP sites) and base modifications sensitive to endonuclease III (5,6-dihydropyrimidine derivatives) were formed in only low amounts. This 'damage profile' and experiments with various scavengers (catalase, superoxide dismutase, deferoxamine, azide, tert-butanol) and D2O as solvent excluded the involvement of hydroxyl radicals and single oxygen in the damage production, but were consistent with a radical mechanism involving bromine radicals. In L1210 mouse leukemia cells and LLC-PK1 porcine kidney cells, KBrO3 alone gave rise to a DNA damage profile similar to that observed after treatment of cell-free DNA with KBrO3 plus GSH, i.e. base modifications sensitive to Fpg protein were formed in high excess of all other lesions quantified. In LLC-PK1 cells (derived from the target organ of KBrO3-induced carcinogenesis) the level of DNA damage was twice that in the L1210 cells. DNA damage was partially prevented by depletion of intracellular GSH with diethylmaleate, indicating that GSH played an activating role in the cells similar to that seen under cell-free conditions. The Fpg-sensitive base modifications induced by KBrO3 were repaired with only moderate efficiency (38 +/- 10% of the lesions were still present after 18 h in full medium) under conditions that did not influence cell proliferation.

Animals↗
PubMed1995

111In-octreotide imaging in patients with long-standing Graves' ophthalmopathy.

The aim of this study was to examine patients with long-standing Graves' ophthalmopathy using 111In-octreotide scintigraphy. Sixteen patients with inactive ophthalmopathy of up to 114 months duration and 14 normals were investigated for 48 h following an injection of 200 MBq 111In-octreotide. No significant tracer accumulation in the orbital region could be identified in any of the patients with long-standing Graves' ophthalmopathy. The orbit to brain (O/B) ratios after 24 and 48 h were 2.39 +/- 0.36 and 2.15 +/- 0.44 versus 2.17 +/- 0.33 and 2.20 +/- 0.37 for the patients and normals, respectively (N.S.). 111In-octreotide accumulation in ophthalmopathy described in the literature may thus be a passing event limited to its active stage, which is consistent with the concept of imaging a lymphocytic infiltration. In this study, the lack of accumulation of 111In-octreotide in the orbital region during the inactive stage demonstrates an absence of somatostatin receptors in orbital tissue itself. Thus, in patients with inactive Graves' ophthalmopathy, there is no basis for a diagnostic approach with somatostatin.

Case-Control Studies↗
PubMed1995

Repair of ultraviolet B and singlet oxygen-induced DNA damage in xeroderma pigmentosum cells.

Ultraviolet B (UVB) (290-320 nm) is capable of damaging the DNA molecule directly by generating predominantly pyrimidine dimers. UVA (320-400 nm) does not alter the DNA molecule directly. However, when it is absorbed by cellular photosensitizers, it can damage the DNA molecule indirectly, e.g., by mediation of singlet oxygen, generating predominantly 8-hydroxyguanine. These indirect effects have been implicated in the mutagenic, genotoxic, and carcinogenic effects of UVA. To study the processing of directly and indirectly UV-induced DNA damage in intact, DNA-repair-proficient and -deficient human cells, we used the replicating plasmid pRSVcat, either irradiated with up to 10 kJ/m2 UVB or treated with the photosensitizer methylene blue plus visible light (which generates singlet oxygen). These treated plasmids were introduced into lymphoblast lines from normal donors or from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and variant. DNA repair was assessed by measuring activity of reactivated chloramphenicol-acetyl-transferase enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 d. As expected, the repair of UVB-induced DNA damage was reduced in all XP cell lines, and the degree varied with the complementation group. XP-A, -D, -E, and -variant cells were normally efficient in the repair of singlet oxygen-induced DNA damage. Only three of four XP-C cell lines showed a markedly reduced repair of these lesions. This indicates differential DNA-repair pathways for directly and indirectly UV-induced DNA damage in human cells and suggests that both may be affected in XP-C.

DNA Damage↗
PubMed1995

Assessment of genotoxicity and mutagenicity of 1,2-dioxetanes in human cells using a plasmid shuttle vector.

1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds on thermal decomposition. They cause photochemical and photobiological transformations in the dark. In order to study the genotoxicity and mutagenicity of 1,2-dioxetanes, the replicating shuttle vector pZ189 was damaged with 3,3,4-trimethyl-1,2-dioxetane (TrMD) or 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD) in vitro and subsequently transfected into normal human lymphoblasts. We found a dose-dependent increase of genotoxicity (decrease of plasmid survival) and increase of mutation frequency with both dioxetanes. However, TrMD was less mutagenic than HTMD at similar genotoxicity. Sequence analysis of the supF gene revealed more point mutations with TrMD and 100% with HTMD were G:C to T:A and G:C to C:G transversions. These are the typical mutations following 7,8-dihydro-8-oxoguanine (8-oxo-G) formation, the main DNA lesion induced by TrMD and HTMD. Only with TrMD we found 5.4% G:C to A:T transitions, probably reflecting the more pronounced ability of TrMD to form some pyrimidine dimers. Our results indicate that 8-oxo-G is also the most relevant modification in in vivo mutagenesis.

Base Sequence↗
PubMed1994

Recognition of oxidized abasic sites by repair endonucleases.

The recognition of 'regular' and 'oxidized' sites of base loss (AP sites) in DNA by various AP endonucleases was compared. Model substrates with regular AP sites (resulting from mere hydrolysis of the glycosylic bond) were produced by damaging bacteriophage PM2 DNA by exposure to low pH; those with AP sites oxidized at the C-4'- and C-1'-position of the sugar moiety by exposure to Fe(III)-bleomycin in the presence of H2O2 and to Cu(II)-phenanthroline in the presence of H2O2 and ethanol, respectively. The results confirmed that AP sites-together with single-strand breaks-are indeed the predominant type of DNA modification in all three cases. For the recognition of 4'-oxidized AP sites, a 400-fold higher concentration of Escherichia coli exonuclease III and between 5-fold and 50-fold higher concentrations of bacteriophage T4 endonuclease V, E. coli endonuclease III and E. coli FPG protein were required than for the recognition of regular AP sites. In contrast, the recognition of 4'-oxidized AP sites by E. coli endonuclease IV was effected by 4-fold lower concentrations than needed for regular AP sites. 1'-oxidized AP sites (generated by activated Cu(II)-phenanthroline) were recognized by endonuclease IV and exonuclease III only slightly (3-fold and 13-fold, respectively) less efficiently than regular AP sites. In contrast, there was virtually no recognition of 1'-oxidized AP sites by the enzymes which cleave at the 3' side of AP sites (T4 endonuclease V, endonuclease III and FPG protein). The described differences were exploited for the analysis of the DNA damage induced by hydroxyl radicals, generated by ionizing radiation or Fe(III)-nitrilotriacetate in the presence of H2O2. The results indicate that both regular and 1'-oxidized AP sites represent only minor fractions of the AP sites induced by hydroxyl radicals.

Bleomycin↗