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PubMed1995

Differences in basal and induced DNA single-strand breaks between human peripheral monocytes and lymphocytes.

The aim of this study was to compare the susceptibility of peripheral monocytes and lymphocytes to oxidant-induced DNA single-strand breaks (SSB). DNA damage was assessed by the alkaline single-cell gel electrophoresis (SCGE) assay. Total peripheral mononuclear leukocytes (PML), PML enriched in lymphocytes and PML enriched in monocytes were used. The basal rate of SSB was measured after in vitro incubation of cells for 1 h in phosphate-buffered saline, and the induced rate after incubation in 10 microM or 50 microM H2O2. Incubation was performed at 4 degrees C to limit the possible influence of DNA repair. Lymphocyte-enriched PML were obtained after adhesion of the monocytes to tissue-culture treated plastic, and monocyte-enriched PML by removal of monocytes from the plastic through trypsin. In all samples, cell differentiation was performed using an immunofluorescence technique with antibodies against T- and B-lymphocytes and cytospin preparations. The rate of SSB was determined by visual scoring according to 6 predefined categories of DNA damage and was expressed as mean score (range 0-500) per 100 cells. There was a linear relationship between the percentage of lymphocytes in the samples and the basal rate of SSB (p < 0.001, slope 0.67 score units per %). The same was true for induced DNA damage after incubation in 10 microM H2O2 (p < 0.001, slope 3.80 score units per %) or 50 microM H2O2 (p < 0.001, slope 3.22 score units per %). These regression analyses revealed a 2.9-fold greater rate of basal DNA damage in lymphocytes compared to monocytes and an 11.3-fold greater rate for the damage induced by 10 microM H2O2. We conclude that there are marked differences in the rate of basal and induced SSB between lymphocytes and monocytes, suggesting differences in antioxidant capacity between the two cell populations. These findings indicate that the assessment of SSB for biomonitoring and genotoxicity testing using PML has to take into account possible changes in cellular composition.

Adult↗
PubMed1991

Doxorubicin and m-AMSA induced DNA damage in blast cells from AML patients.

We investigated m-AMSA or doxorubicin (Dx) induced DNA single-strand breaks (DNA-SSB) in myeloid leukemia cells obtained from 8 adult patients suffering from AML. Highly purified AML cells were stimulated to proliferate with the addition of the appropriate growth factor (GCT) and exposed to different concentrations of m-AMSA or Dx for 1 or 4 h, respectively. DNA-SSB were determined by alkaline elution techniques. Either the kinetics or the amounts of DNA-SSB caused by both topoisomerase II inhibitors were variable among different cases. By increasing m-AMSA concentrations there was a concomitant increase in DNA-SSB up to a plateau at the highest concentrations. Dx induced DNA-SSB followed a bell shape curve with a decrease in the number of breaks at the highest concentrations that was evident in most cases. The interindividual variability of Dx-induced DNA-SSB was not correlated with intracellular Dx concentrations as assessed by flow cytometry. No correlation was evident between the amount of DNA breaks induced by m-AMSA and that induced by Dx. These data suggest that AML cells derived from different patients are not necessarily cross-sensitive or cross-resistant to topoisomerase II inhibitors with different chemical structures such as amsacrine or anthracyclines.

Adult↗
PubMed1989

Factors influencing the induction of DNA single strand breaks in rats by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce lipid peroxidation and DNA damage in rat hepatic nuclei was investigated. In addition, the role of iron in hepatic DNA damage was examined. Female Sprague-Dawley rats were treated with a single dose of 25--100 micrograms TCDD/kg orally, and sacrificed 3-14 days after treatment. Liver nuclei were isolated, and DNA single strand breaks (DNA-SSB) and lipid peroxidation were determined. Lipid peroxidation was assessed by measuring the content and production of thiobarbituric acid reactive substances (TBARS) while DNA-SSB were determined by the alkaline elution technique. The results demonstrate that TCDD dose and time-dependent increases in hepatic nuclear TBARS content and production occur in conjunction with an increase in DNA-SSB. The administration of the dithiolthione antioxidant oltipraz (30 mg/kg/day for 10 days) resulted in a significant decrease (47%) in the incidence of TCDD-induced DNA-SSB. Clofibrate administration (100 mg/kg/day for 12 days) and pair feeding had no effect on TCDD-induced DNA-SSB. The incubation of hepatic microsomes and mitochondria from TCDD-treated rats with nuclei from untreated animals for one hr at 37 degrees C resulted in enhanced DNA damage which was abolished by the addition of 0.10 mM desferrioxamine (DFX). Incubation with cytosol had no significant effect. Incubation of 0.10 mM Fe2+ or Fe3+ with isolated hepatic nuclei from untreated rats produced significant increases in DNA-SSB, which were abolished by the addition of 0.10 mM DFX to the incubation medium. TCDD may produce an increased bioavailability of iron which leads to enhanced DNA single strand breaks and lipid peroxidation in hepatic nuclei.

Animals↗
PubMed1988

Damage and repair of mouse lung DNA induced by 1-nitropyrene.

The induction of DNA single-strand breaks (SSB), the repair of SSB, and the role of cell turnover in the removal of SSB were determined in mouse lung following intratracheal instillation of 1-nitropyrene (1-NP). Cellular DNA was prelabeled with [3H]thymidine in neonate and adult mice. 1-NP was administered to labeled neonate mice after they became adults and to the adult mice that were labeled when adults. 1-NP induced a dose-related increase in SSB (10-22 times control rate) as early as 2 h after 1-NP administration. The mice labeled as neonates were more sensitive to SSB induction by 1-NP and had a faster repair rate than mice labeled when adults. By one week after 1-NP administration, the levels of SSB in both groups of mice were similar to controls. The half-lives for DNA turnover in mice prelabeled as neonates and in mice prelabeled when adults were approximately 22 and approximately 9 days, respectively, at the time of 1-NP treatments. These data indicate that both highly proliferative cell populations and populations with lower rates of proliferation are amenable to 1-NP-induced DNA lesions. The rate of cell DNA turnover suggests that active DNA repair processes are involved in the removal of DNA lesions. The slower rate of DNA repair coupled with a relatively high rate of cell division in the rapid proliferative cells suggest that these cells may be involved in the induction of cancer in animals after 1-NP administration.

Age Factors↗
PubMed1995

Changes in lymphocyte single strand breakage and liver function of workers exposed to vinyl chloride monomer.

Vinyl chloride monomer (VCM) is a suspected human carcinogen. Its metabolite, chloroethylene epoxide, is able to alkylate the DNA molecule and to produce single strand breakage (SSB). A total of 244 workers from 4 polyvinyl chloride (PVC) manufacturing factories were recruited to assess the SSB of their peripheral lymphocyte DNA. The method of alkaline unwinding and hydroxyapatite chromatography was used to detect and calculate frequencies of SSB. In addition, hepatitis B and C markers and the liver function of the workers were also examined. The worker's cumulative exposures to VCM were retrospectively constructed from the current monitoring data and each worker's job history. Multiple linear regression models were constructed to predict the worker's level of SSB and liver functions based on various exposure indices and variables, such as age, sex, smoking, drinking, and hepatitis markers. The results showed that current smoking and drinking status, and the presence of VCM exposures on the previous day were 3 major determinants of the level of SSB. Among the liver function tests, only gamma-glutamyl transpeptidase (GGT) was associated with current VCM exposures. In contrast, aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were mainly affected by the presence of hepatitis B surface antigen (HBsAg) or anti-hepatitis C virus (anti-HCV). We conclude that GGT should be considered to be included in the regular health screening of VCM workers, and that the SSB method may not be suitable for long-term monitoring of cumulative exposure because of the quick DNA repair mechanism in humans.

Adult↗
PubMed1992

Repair of X-ray induced DNA strand damage by isolated rat splenic lymphocytes.

Although a number of chemicals can alter DNA repair function, little is known about the effect of chronic, low dose exposure to environmental agents on DNA repair capacity. Lymphocytes provide a potential target population to study the effects of chronic exposures to low doses of toxic chemicals since they are an easily obtainable cell population. Prior to investigating the repair capacity of chemically exposed lymphocytes, the repair by chemically naive lymphocytes has been characterized. In the present study, the DNA repair capacity of isolated rat lymphocytes was characterized. The capacity of these cells to repair single-strand DNA breaks (SSB) was determined after in vitro treatments with X-rays. The effect of in vitro exposure to 3-aminobenzamide (3-AB) on DNA repair capacity was also assessed. The levels of induced SSB and their repair were determined using the alkaline elution technique. Splenic lymphocytes were isolated and placed in culture medium 18 h prior to assessment of repair capacity, but were not stimulated with mitogens. A dose-dependent increase in SSB was observed following exposure of lymphocytes to 300 or 600 rad. The rate of SSB repair was analyzed after a dose of 400 rad. Approximately 80% of the DNA strand break repair was completed within 60 min. The half-time for repair of these lesions by lymphocytes was determined to be 21.3 min. Exposure to 3-AB resulted in a decrease in the rate of repair of the X-ray-induced strand breakage. Although no SSB were detected at the end of a 1-h 3-AB treatment of non-irradiated cells, significant accumulation of SSB was observed after a 2-h treatment. The characterization of DNA repair in rat lymphocytes following in vitro exposure to X-rays will allow us to investigate the effects of chronic, in vivo toxicant exposure on the capacity of isolated lymphocytes to repair DNA damage produced by X-rays.

Animals↗
PubMed1992

HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.

Bacterial Proteins↗
PubMed2005

The effect of the air-blowing step on the technique sensitivity of four different adhesive systems.

PURPOSE: To evaluate the technique sensitivity of four different adhesive systems using different air-blowing pressure. METHODS: Four adhesive systems were employed: Clearfil SE Bond [SE] (Kuraray, Japan), G-Bond [GB] (GC Corporation, Japan), Adper Prompt L-Pop [LP] (3M ESPE, USA) and an experimental adhesive, SSB-200 [SSB] (Kuraray, Japan). Twenty-four extracted molars were used. After grinding the coronal enamel surface, the teeth were divided into two equal groups. The first group's teeth were randomly assigned for bonding with the different adhesives using gentle air-blowing (g). For the teeth of the second group, the four adhesive systems were applied using strong air-blowing (s). After storage overnight in 37 degrees C water, the bonded specimens were sectioned into sticks (1 mm x 1 mm wide), which were subjected to microtensile bond strength testing (microTBS) at a crosshead speed of 1 mm/min. The load at failure of each specimen was recorded and the data were analyzed by one-way ANOVA and Tukey HSD tests. The surfaces of the fractured specimens were observed using SEM to determine the failure mode. RESULTS: The results of the microTBS test showed that the highest bond strengths tended to be with SE for both gentle and strong air-blowing, and the significantly lowest for SSB with strong air streaming. Comparing the two techniques, significant differences were noted only for SSB-200 (P < 0.05). For each material, the SEM evaluation did not show distinct differences in the nature of the fractures between the two techniques, except for SSB-200. CONCLUSIONS: The adhesives tested are not technique sensitive, except SSB-200, with regards to the air-blowing step.

Air↗
PubMed1989

Triplet state properties of tryptophan residues in complexes of mutated Escherichia coli single-stranded DNA binding proteins with single-stranded polynucleotides.

Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed.

DNA-Binding Proteins↗
PubMed2001

SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay).

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.

Ascorbic Acid↗
PubMed2002

Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus-expression and purification.

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. We report here the identification, expression, and purification of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and T. aquaticus. The nucleotide (nt) sequence revealed that T. thermophilus SSB (TthSSB) and T. aquaticus (TaqSSB) consist of 264 and 266 amino acids, respectively, and have a molecular weight of 29.87 and 30.03kDa, respectively. The homology between these protein, is very high-82% identity and 90% similarity. They are the largest known prokaryotic SSB proteins. TthSSB and TaqSSB monomers have two putative ssDNA-binding sequences: N-terminal (located in the region from amino acids 1 to 123) and C-terminal (located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively). PCR-derived DNA fragment containing the complete structural gene for TthSSB or TaqSSB protein was cloned into an expression vector. The clones expressing SSB-like proteins were selected and cloned DNA fragments were verified to be authentic by sequencing several clones. The purification was carried out using reduction of contamination by the host protein with heat treatment, followed by QAE-cellulose and ssDNA-cellulose column chromatography. We found our expression and purification system to be quite convenient and efficient, and will use it for production of thermostable SSB-proteins for crystallography study. We have applied the use of TthSSB and TaqSSB protein to increase the amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving the PCR efficiency.

Amino Acid Sequence↗
PubMed1999

An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers.

A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.

Adult↗
PubMed1987

A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions.

We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.

Bacterial Proteins↗
PubMed1990

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy.

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.

Bacterial Proteins↗
PubMed2006

Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase.

Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone (TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s(-1), respectively). Thus, Asp298 plays important roles not only in the alpha-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.

Amine Oxidase (Copper-Containing)↗
PubMed2002

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation.

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.

DNA Damage↗
PubMed2005

Correlation of free radical yields with strand break yields produced in plasmid DNA by the direct effect of ionizing radiation.

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(Sigmafr), (ii) radicals trapped on the DNA sugar, Gsugar(fr), (iii) prompt single strand breaks, Gprompt(ssb), (iv) total single strand breaks, Gtotal(ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(fr) were measured by EPR applied to films of pEC (10,810 bp) and pUC18 (2686 bp) plasmids hydrated to Gamma = 22 mol of water/nucleotide and X-irradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(fr) = 0.71 +/- 0.02 and 0.61 +/- 0.01 micromol/J, Gtotal(ssb) = 0.09 +/- 0.01 and 0.14 +/- 0.01 micromol/J, G(dsb) = 0.010 +/- 0.001 and 0.006 +/- 0.001 micromol/J, and Gtota)(ssb)/G(dsb) approximately 9 and approximately 20. Surprisingly, Gsugar(fr) approximately 0.06 mumol/J for pUC18 films, less than half of Gtotal(ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.

DNA↗
PubMed1998

Probing the mechanism of the carcinogenic activation of N-nitrosodiethanolamine with deuterium isotope effects: in vivo induction of DNA single-strand breaks and related in vitro assays.

A series of bioassays, including in vivo induction of DNA single-strand breaks (SSB) and cytotoxicity in cytochrome P450 2E1-transfected cells, were utilized with N-nitrosodiethanolamine (NDELA), its deuterated isotopomers (alpha-D4NDELA and beta-D4NDELA), N-nitroso-2-hydroxymorpholine (NHMOR), and two of its deuterated isotopomers (2-D-NHMOR and 5,5-D2-NHMOR) to probe the mechanism of carcinogenic activation of NDELA and the role of its metabolite NHMOR. DNA samples, taken from the livers of male Wistar rats 4 h after the administration of NDELA, exhibited dose-dependent DNA SSB levels over the range of 0.08-0.75 mmol/kg (body weight), with the greatest SSB level at the highest dose. Deuterium isotope effects on DNA SSB levels were inversely dependent on dose: alpha-D4NDELA, 3. 22-1.37; and beta-D4NDELA, 1.38-0.79. At the lowest dose of 0.15 mmol/kg (body weight), 5,5-D2-NHMOR gave an isotope effect for DNA SSB of 2.8 while that for 2-D-NHMOR was 0.7. NDELA and beta-D4NDELA were equally cytotoxic to human P450 2E1-transfected V79 Chinese hamster cells, while alpha-D4NDELA was not. Significant DNA SSB levels were observed in these cells for NDELA and beta-D4NDELA but not for alpha-D4NDELA. A kinetic deuterium isotope effect of 2.6 for Vmax/Km was observed for the horse liver alcohol dehydrogenase-mediated oxidation of beta-D4NDELA to NHMOR, while kH/kD for alpha-D4NDELA was 1.05. These data provide the first definitive evidence for the activation of NDELA by a pathway involving the scission of the alpha-CH bond and are consistent with P450 2E1-mediated alpha-hydroxylation of NDELA producing the corresponding reactive alpha-hydroxynitrosamine.

Animals↗