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PubMed1985

Repair of DNA single-strand breaks in X-irradiated yeast. I. Use of the DNA-unwinding method to measure DNA strand breaks.

The DNA-unwinding method developed by Ahnström and his coworkers to measure DNA strand breaks in mammalian cells was used to measure single-strand breaks (SSB) in the DNA of intact yeast cells. DNA unwinding, which took place inside the rigid cell wall of yeast, was investigated as a function of time, radiation dose, and of pH and salt concentration of the alkaline solution. After DNA unwinding had taken place, the cell wall was destroyed by partial enzymatic digestion and sonication in the presence of detergents. Fragments of single- and double-stranded DNA were separated using hydroxylapatite chromatography. In this way the most suitable conditions for DNA unwinding within the cell wall were established. The results show that SSB and double-strand breaks (DSB) give rise to different kinetics of DNA unwinding.

DNA Repair↗

PubMed1989

Investigations on organ-specific metabolism and genotoxic effects of the urinary bladder carcinogen N-nitrosobutyl-3-carboxypropylamine (BCPN) and its analogs N-nitrosodibutylamine (NDBA) and N-nitrosobutyl-4-hydroxybutylamine (4-OH-NDBA).

N-Nitrosodibutylamine (NDBA) and its omega-oxidized metabolites N-nitrosobutyl-4-hydroxybutylamine (4-OH-NDBA) and N-nitrosobutyl-3-carboxypropylamine (BCPN) are potent urinary bladder carcinogens. To study putative organ specific activation of BCPN, its alpha-oxidation by liver and urinary bladder microsomal fractions was investigated in comparison to NDBA and 4-OH-NDBA. Additionally, induction of DNA single strand breaks (SSB) was monitored in hepatocytes and in a human lymphoblastoid cell line (Namalva) in the presence and absence of external metabolic activation, including N-nitroso-t-butyl-n-butylamine as a negative control. BCPN was alpha-hydroxylated and dealkylated at both alkyl chains in small rates (about 1 nmol x mg protein-1 x 60 min-1) by microsomes from rat liver and pig urinary bladder epithelium. NDBA and 4-OH-NDBA were dealkylated at similarly low rates by pig urinary bladder microsomes, in strong contrast to the high debutylation rates observed for rat liver microsomes. Correspondingly, SSB induction by NDBA and 4-OH-NDBA was observed in Namalva cells with NDBA and 4-OH-NDBA in the presence of PB-induced rat liver microsomes but not with urinary bladder microsomes or without external activation. BCPN did not induce DNA-damage in Namalva cells (with or without external activation) or in rat hepatocytes. Significant induction of sister chromatid exchanges (SCEs) and micronuclei, however, was observed in Namalva cells after incubation with NDBA and BCPN. Our data suggest activation of BCPN via alpha-oxidation in the urinary bladder, even though activation rate in-vitro is so low that a positive response is not detectable by several short-term tests.

Animals↗

PubMedDate not supplied

Biochemical studies of homologous and nonhomologous recombination in human cells.

Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro. Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein. Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold. Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons. We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa. Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells. A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.

Base Sequence↗

PubMedDate not supplied

Characterization of recF suppressors in Bacillus subtilis.

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.

Bacillus subtilis↗

PubMed1993

A comparative study of Scatchard-type and linear lattice models for the analysis of EPR competition experiments with spin-labeled nucleic acids and single-strand binding proteins.

An EPR competition formalism is developed which provides relative affinities of proteins for nucleic acids. Two models for analyzing protein-nucleic acid interactions, one assuming independent binding sites (Model 1) and the other considering site overlap (Model 2), are examined with respect to their validity and limitations. The models are employed to derive affinity ratio relationships which are used to calculate the relative affinities of gene 32, gene 5, and SSB proteins for various nucleic acids. It is determined that although Model 2 must be used when determining absolute binding constants, by taking the ratio of binding constants the site overlap becomes unimportant under conditions of moderate to high cooperativity and relatively small site size. This allows Model 1 to considerably simplify binding analyses. Both models are applied to the single-strand binding proteins of bacteriophage T4 gene 32, bacteriophage fd gene 5, and the Escherichia coli ssb gene, and the results are compared.

Allosteric Regulation↗

PubMed1992

Multiple effects of 3-aminobenzamide on DNA damage induced by cisplatin (DDP) in DDP-sensitive and -resistant rat ovarian tumor cell lines.

To investigate the mechanisms by which 3-aminobenzamide (3AB) reverses cisplatin (DDP) resistance in a rat ovarian tumor cell line, the effects of 3AB on DDP-induced DNA damage and repair were kinetically determined over a post-exposure period of 48 h. DNA single strand breaks (SSB) occurred maximally 12 h and 24 h following DDP exposure in DDP-resistant (O-342/DDP) and -sensitive (O-342) rat ovarian tumor cells, respectively. 3AB, present during and after the exposure, significantly increased SSB formation by DDP at 24 h (P < 0.02) and 48 h (P < 0.01) in O-342/DDP cells. To a lesser extent (P > 0.05), a similar tendency was also observed in O-342 cells. Formation of DNA interstrand cross-links (ISCL) by DDP reached a maximum by 12 h in either O-342 or O-342/DDP cells, but in the resistant cells they were both much lower and more rapidly removed. 3AB decreased ISCL in the sensitive cells at 12 h and thereafter with a maximum at 24 h (P < 0.05), while in the resistant cells the same treatment decreased ISCL at 12 h, had no effect at 24 h and increased ISCL at 48 h following DDP treatment. Therefore, it is concluded that 3AB has multiple effects on DNA damage and repair induced by DDP in both cell lines and increase of DNA-ISCL by 3AB at 48 h after the exposure in O-342/DDP cells might be related to its chemosensitizing effect in this line.

Animals↗

PubMed1992

Mechanisms of DNA damage induced in rat hepatocytes by quinones.

Hydroquinone (HQ), catechol (CAT), duroquinone (DQ) and resorcinol (RES) have been shown to induce single-strand breaks (SSB) in DNA in isolated hepatocytes using the alkaline elution assay. The relative capacities of these agents to induce SSB were shown to be: DQ = HQ much greater than CAT greater than RES. Preincubation of hepatocytes with the Ca(2+)-chelator Quin-2 AM reduced the level of DNA damage to a great extent, as did treatment with DQ and RES, and, to a lesser extent, treatment with HQ and CAT. Preincubation with an inhibitor of poly(ADP-ribose) polymerase, 3-amino-benzamide, did not influence the extent of DNA damage caused by DQ and RES, but increased the damage caused by HQ and CAT. The conclusion of the present investigation was that the dominating reaction of HQ and CAT, with DNA is an arylation, whereas DQ and RES induce DNA damage by oxidative stress.

Animals↗

PubMed1994

Vitamin E acts as a useful chemopreventive agent to reduce spontaneous lung tumorigenesis in mice.

The present study was designed to evaluate whether vitamin E could be a useful chemopreventive agent to reduce spontaneous lung tumorigenesis in mice. Starting at 6 weeks of age, groups were divided into three groups, i.e. A/J mice fed a control diet (A/J control), A/J mice fed a vitamin E-supplemented diet (A/J vitamin E) and ddY mice fed a control diet (ddY control). At the 28th experimental week, nuclear NADPH-driven active oxygen generation, thiobarbituric acid reactive substances (TBARS) and DNA single strand breaks (DNA-SSB) in A/J mice fed a control diet were significantly higher than those in the ddY control group. A/J mice fed Vitamin E for 28 weeks could decrease the levels of TBARS and DNA-SSB with a significant difference, as compared with those in A/J control mice. The nuclear alpha-tocopherol levels in A/J controls were significantly lower than those in ddY controls, on the contrary, the vitamin feeding to A/J mice increased nuclear alpha-tocopherol levels more than that in the ddY controls. At the 40th experimental week, lung tumor incidence and tumor multiplicity (percentage of mice with tumors) in A/J controls were reduced and brought close to those in ddY control mice by vitamin E. Then the alpha-tocopherol level in plasma of A/J controls was significantly lower than the level in plasma of ddY controls, and the level in tumor-bearing mice in A/J controls also showed a lower level with significant difference as compared to that in non-tumor-bearing mice of A/J controls. These results suggest that the difference in susceptibility to spontaneous lung tumorigenesis between A/J and ddY mice partly depends on the difference of oxidative stress on the pulmonary nuclei, and vitamin E can act as a useful chemopreventive agent to reduce spontaneous lung tumorigenesis in mice.

Animals↗

PubMed1996

Anatomical distribution of somatostatin receptors in the brainstem of the human fetus.

The distribution of somatostatin binding sites was studied in the pons and medulla oblongata of three human fetuses (gestional ages 26, 28 and 30 weeks). The study was carried out by in vitro quantitative autoradiography using either [125I-Tyr0,D-Trp8]somatostatin-14 or [125I-Tyr11]somatostatin-14 as radioligands. Somatostatin binding sites were observed in a number of nuclei subserving sensory, motor or integrative functions within the pons and medulla. In addition, discrete tracts also contained significant amounts of binding sites. Among structures involved in sensory processes, a high density of binding sites (40-60 fmol/ mg wet tissue) was measured in the dorsal cochlear nucleus and in the nucleus tractus spinalis trigemini caudalis. Moderate to high levels of binding sites (30-40 fmol/mg wet tissue) were detected in the other sensory cranial nerve nuclei. A moderate density of sites (15-30 fmol/mg wet tissue) was measured in most motor nuclei, the highest concentrations being observed in the dorsal motor nucleus of the vagus nerve, the facial nucleus, the hypoglossal nucleus and the nucleus ambiguus. The griseum pontis and the nucleus corporis pontobulbaris contained very high (> 60 fmol/mg wet tissue) and high concentrations of somatostatin binding sites, respectively, while the other relay nuclei contained low to moderate levels of binding. In monoaminergic nuclei, very high and moderate to high concentrations of somatostatin binding sites were measured in the nucleus locus coeruleus and in its dorsal subnucleus, respectively. Moderate densities of sites were detected in the ventral subnucleus of the nucleus locus coeruleus and in the different parts of the raphe. In the white matter, low levels of binding were measured in the inferior cerebellar peduncle, the lateral and median lemnisci and the tractus solitarius. Conversely, moderate to high concentrations of somatostatin binding sites were measured in the median and superior cerebellar peduncles. The pyramis contained a very high density of recognition sites. A marked heterogeneity in the density of binding sites was observed within a few structures particularly in the medial accessory olivary of nucleus and the medial longitudinal fasciculus. Selective ligands were used to determine the pharmacological profile of the [Tyr11]somatostatin-14 binding sites in various brainstem regions. In the dorsal cochlear nucleus and the pyramis, all somatostatin binding sites belonged to the SSA subtype. Conversely, in the lateral paragigantocellular nucleus, all somatostatin binding sites belonged to the SSB subtype. The other regions studied contained various proportions of SSA and SSB subtypes. In conclusion, the present study shows that high concentrations of somatostatin receptors are present in many regions of the human fetus brainstem. These data support the concept that somatostatin could be involved in the maturation of brain structures.

Age Factors↗

PubMed1986

Protective effects of 2-[(aminopropyl)amino] ethanethiol against bleomycin and nitrogen mustard-induced mutagenicity in V79 cells.

This study examines the effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on bleomycin (BLM) and nitrogen mustard- (HN2) induced cytotoxicity, DNA damage, and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells. The anti-mutagenic effect of WR-1065 on cis-diamminedichloroplatinum (cis-DDP) and radiation- (XRT) induced HGPRT mutations was also evaluated for comparative purposes. WR-1065 (4 mM) was added prior to exposure of cells to therapy agents. All exposure times were 30 min. and both cell survival and mutagenesis were assayed. WR-1065 was effective in protecting against both effects. The induction of mutants corrected for background by BLM, HN2, cis-DDP, or XRT was linear in all cases. Mutation frequencies without WR-1065 were 78 X 10(-6) per unit BLM, 66 X 10(-7) per microgram HN2, 25 X 10(-7) per microgram cis-DDP; and 87 X 10(-7) per Gy of XRT. With WR-1065, these were reduced to 37 X 10(-6) per unit BLM, 40 X 10(-7) per microgram HN2, 1 X 10(-7) per microgram cis-DDP, and 44 X 10(-7) per Gy of XRT. Mutation protection factors (MPF), a ratio of the corresponding slopes of the mutation induction curves, with and without WR-1065 were: BLM, MPF = 2.8; HN2, MPF = 3.4; cis-DDP, MPF = 7.1; and XRT, MPF = 5.1. Single-strand-break (SSB) formation in DNA by BLM or HN2, assayed by alkaline elution, was protected against by WR-1065. WR-1065 did not induce SSB in control cells. The reduction of the mutagenic effects of agents used in radiation and chemotherapy by radioprotectors may be an important additional benefit for consideration in their use in cancer treatment.

Animals↗

PubMed1986

Mechanisms of DNA repair and their potential modification for radiotherapy.

The potentiation of radiation damage, which can be accomplished by the inhibition of repair, is estimated from published studies of repair deficient mutants. Sensitization factors as high as 10 have been achieved. Because it has previously been suggested that the most probable lethal lesion is a DNA double strand break (DSB), it is not surprising that cells deficient in repairing this type of damage are the most radiosensitive. The structures of DNA DSBs and other Locally Multiply Damaged Sites (LMDS) (involving both single strand breaks (SSB) and base damage sites) are reviewed, together with the processes by which cells may attempt to repair these lesions. Repair processes occur in competition with damage fixation, again, mechanisms of damage fixation are predicted from studies in model systems. A strategy for inhibiting the repair processes is devised that consists of holding the first SSB constituent of the LMDS open by repairing in the presence of deoxynucleoside analogues, such as ara-C, so that there is a higher probability of the formation of a DSB upon cleavage of the second site (on the other strand) by hydrolysis of a labile bond or by endonuclease cleavage of a base damaged site. To achieve preferential sensitization of tumor vs. normal tissue it may be possible to take advantage of the deficiency in alkaline phosphatase in tumor vs. normal vasculature, that is, in analogy with treatment with WR-2721. The deoxynucleoside analogue would be delivered together with the phosphate ester (deoxynucleotide) of the correct deoxynucleoside, for example, ara-C, in the presence of deoxycytidine monophosphate (dCMP). Higher alkaline phosphatase levels in normal tissue capillaries would hydrolyse the dCMP to deoxycytidine, which competes effectively with ara-C in repair replication.

Animals↗

PubMed1988

A plasmid vector system for the expression of a triprotein consisting of beta-galactosidase, a collagenase recognition site and a foreign gene product.

A plasmid-cloning vector system has been constructed which allows the production of fusion proteins with beta-galactosidase at the N terminus, followed by a recognition sequence for the site-specific protease, collagenase, and the foreign protein at the C terminus. A multicloning site allows the insertion of foreign genes in any translational reading frame. Fusion proteins were isolated by affinity chromatography on APTG-Sepharose. The foreign protein was released from the fusion product by collagenase cleavage. The vector was successfully utilized for the production of Escherichia coli single-stranded (ss) DNA-binding protein (SSB protein). The proteolytically released SSB protein resisted elution from an ss DNA-cellulose column with 1 M NaCl.

Bacterial Proteins↗

PubMed1995

Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats.

Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Recent studies have demonstrated that sodium dichromate [Cr(VI)] induces greater oxidative stress as compared with Cr(III), as indicated by the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes, and enhanced excretion of urinary lipid metabolites and hepatic DNA-single strand breaks (SSB) following acute oral administration of Cr(III) and Cr(VI). We have therefore examined the chronic effects of sodium dichromate dihydrate [Cr(VI); 10 mg (33.56 mumol)/kg/day] on hepatic mitochondrial and microsomal lipid peroxidation, enhanced excretion of urinary lipid metabolites including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), acetone (ACON) and propionaldehyde (PROP), and hepatic DNA damage over a period of 90 days. The maximal increases in hepatic lipid peroxidation and DNA damage were observed at approximately 45 days of treatment. Maximum increases in the urinary excretion of MDA, FA, ACT, ACON and PROP were 3.2-, 2.6-, 4.1-, 3.3- and 2.1-fold, respectively, while a 5.2-fold increase in DNA-SSB was observed. The results clearly indicate that chronic sodium dichromate administration induces oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of hexavalent chromium.

Acetaldehyde↗

PubMed1991

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.

Adenocarcinoma↗

PubMed1993

Protective effects of antioxidants against endrin-induced hepatic lipid peroxidation, DNA damage, and excretion of urinary lipid metabolites.

Oxidative stress is believed to play a pivotal role in endrin-induced hepatic and neurologic toxicity. Therefore, the effects of the antioxidants vitamin E succinate and ellagic acid have been examined on hepatic lipid peroxidation, DNA single-strand breaks (SSB), and the urinary excretion of lipid metabolites following an acute oral dose of 4.5 mg endrin/kg. Groups of rats were pretreated with 100 mg/kg vitamin E succinate for 3 d followed by 40 mg/kg on day 4, or 6.0 mg ellagic acid/kg for 3 d p.o. followed by 3.0 mg/kg on day 4 or the vehicle. Endrin was administered p.o. on day 4 2 hr after treatment with the antioxidant. All animals were killed 24 h after endrin administration. Vitamin E succinate pretreatment decreased the endrin-induced increase in hepatic mitochondrial and microsomal lipid peroxidation by approximately 60% and 40%, respectively. Ellagic acid pretreatment reduced the endrin-induced increased in mitochondrial and microsomal lipid peroxidation by approximately 76 and 79%, respectively. Both vitamin E succinate and ellagic acid alone produced small but nonsignificant decreases in hepatic mitochondrial and microsomal lipid peroxidation. A 3.3-fold increase in the incidence of hepatic nuclear DNA single-strand breaks was observed 24 h after endrin administration. Pretreatment of rats with vitamin E succinate, vitamin E, and ellagic acid decreased endrin-induced DNA-SSB by approximately 47%, 22%, and 21%, respectively. Pretreatment of rats with vitamin E succinate decreased the endrin-induced increase in the urinary excretion of malondialdehyde, acetaldehyde, formaldehyde, and acetone by approximately 68, 65, 70, and 55%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗

PubMed1995

Protective effects of lazaroid U74389F (16-desmethyl tirilazad) on endrin-induced lipid peroxidation and DNA damage in brain and liver and regional distribution of catalase activity in rat brain.

Endrin, a poly-halogenated cyclic hydrocarbon, induces hepatic lipid peroxidation, modulates calcium homeostasis, decreases membrane fluidity, and increases nuclear DNA damage. Little information is available on the neurotoxicity of endrin. The effects of endrin on lipid peroxidation, DNA damage, and regional distribution of catalase activity were assessed in rat brain and liver 24 h following an acute oral dose of 4.5 mg endrin/kg. Lipid peroxidation associated with whole brain mitochondria increased 2.4-fold, whereas microsomal lipid peroxidation increased 2.8-fold following endrin administration. Lipid peroxidation also increased 2.0-fold both in hepatic mitochondria and microsomes. Catalase activity decreased 24% in the hypothalamus, 23% in the cortex, 38% in the cerebellum, and 11% in the brain stem in response to endrin. A 4.3-fold increase in brain nuclear DNA-single strand breaks (SSB) was observed in endrin-treated rats. Pretreatment of rats intraperitoneally with the lazaroid U74389F (16-desmethyl tirilazad) (10 mg/kg in two doses) attenuated the biochemical consequences of endrin-induced oxidative stress. The administration of U74389F in citrate buffer (pH 3.8) provided better protection than administering the lazaroid in corn oil, decreasing endrin-induced lipid peroxidation by 50-80% and DNA-SSB by approximately 72% in liver and 85% in brain, while ameliorating the suppressed catalase activity. The data suggest an involvement of an oxidative stress in the neurotoxicity and hepatotoxicity induced by endrin, which can be attenuated by the lazaroid U74389F.

Animals↗

PubMed1996

Factors affecting DNA damage caused by lipid hydroperoxides and aldehydes.

Single (SSB) and double strand breaks (DSB) in supercoiled plasmid DNA pBR322 reacted with linoleic acid hydroperoxides (LOOH) were followed by agarose gel electrophoresis to obtain definitive information about factors affecting LOOH interaction with DNA. In water, LOOH induced extensive DSB, which were metal mediated and increased with incubation time. Adventitious metal bound to DNA was sufficient to decompose LOOH to reactive radicals, activity that was not readily inhibited by chelators DTPA and desferrioxamine. Added Fe2+ and Fe3+ increased SSB and DSB, although the effects of Fe2+ were more extensive. Above 100 microM both valences inhibited DNA damage. Strand breakage by LOOH proceeded via lipid alkoxyl and peroxyl radicals. Aldehydic lipid peroxidation products induced strand breaks via oxidation of double bonds, not by reactions of the carbonyl groups. Lipophilic antioxidants BHA, BHT, and alpha-tocopherol were about 20 times more effective than hydrophilic free radical scavengers sodium benzoate, inositol, DMSO, and mannitol in preventing LOOH-induced strand breaks, supporting lipid phase localization of the damage.

Aldehydes↗

PubMed1996

Association between 8-hydroxy-2'-deoxyguanosine formation and DNA strand breaks mediated by copper and iron.

Oxidative DNA damage is involved in diverse biological phenomena and consists of several kinds of lesions, mainly, strand breaks, base modifications, and DNA-protein crosslinking. However, little is known about the existence of a chemical relationship among them or the ratio by which these different types of lesions are produced. In the present study we investigated whether a relationship exists between DNA strand breakage and base modification. We selected cupric [Cu(II)] and ferric [Fe(III)] ions for this study because these transition metals are active catalysts of DNA damage in vivo. Supercoiled plasmid DNA pZ189 was treated with Cu(II) or Fe(III) in the presence of different reducing agents. We measured in each sample both the number of DNA single-strand breaks (SSB) by quantitative electrophoresis and the amount of a modified DNA base, 8-hydroxy-2'-deoxyguanosine (8-OHdG) by HPLC with simultaneous electrochemical (EC) and spectrophotometric detection. The number of DNA SSBs produced was linearly related to the number of 8-OHdG present. The regression of the number of SSBs as a function of the number of 8-OHdG is expressed by the equation [SSBs] = b x [8-OHdG], where b = 1.7, 2.0, 2.7, 1.7, and 9.4, for Cu(II) in the presence of H2O2, L-cysteine and L-ascorbate, and for Fe(III) in the presence of H2O2 and L-ascorbate, respectively. The linear correlation we observed between the production of SSB and 8-OHdG mediated by Fe(III) and by Cu(II) suggests that these products may arise via a common chemical mechanism and could allow an easier and more precise estimation of DNA breakage.

8-Hydroxy-2'-Deoxyguanosine↗