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U.S. Navy Asbestos Medical Surveillance Program B-reading reports were analyzed for variability in identifying pneumoconiosis in 278,944 chest radiographs done from 1990 to 2004 using commercial database and statistical software. Reported presence of pneumoconiosis by 33 B-readers ranged from 1.85 to 32.28%. The reported presence of parenchymal abnormalities ranged from 0.11 to 15.07%; increased profusion (to 1/0 or greater) of small parenchymal opacities ranged from 0.10 to 17.70%; presence of large opacities ranged from 0.00 to 0.67%; and presence of pleural abnormalities ranged from 1.08 to 23.93%. Ranges appear to be decreasing slightly as rates of abnormal findings are diminishing. Differences between U.S. East Coast, West Coast, and midcontinent were not statistically significant. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To study the impact of variability among B-readers on clinical occupational medicine. METHODS: A total of 419,770 B-reading reports from February 1980 to May 2004 in the US Navy Asbestos Medical Surveillance Program were analyzed for changes in category from the previous B-reading on each individual worker. RESULTS: Over 7% of films were categorized as worse (ie, read as going from negative to positive), and over 6% were categorized as better. When profusion categories were reported as different from the previous reading (over 6% of the time), they were more frequently read as 2 or more minor categories worse or better. CONCLUSIONS: Changes from previous B-readings are common, and may have clinical and other implications, which are discussed. B-readings should not be used as the sole basis for determining the presence or absence of pneumoconiosis. [ABSTRACT FROM AUTHOR]

The U.S. Navy Asbestos Medical Surveillance Program is a comprehensive effort to decrease exposure to asbestos, a known health hazard. This study was part of a programmatic review of the Asbestos Medical Surveillance Program database, which included 233,353 radiographic examinations from 1990 to 1999. The initial review focused on incidental findings recorded by B-readers for 23,460 radiographs. Abnormalities reported included bullae (0.68%), cancer (0.56%), cardiac size/ shape abnormalities (1.36%), emphysema (0.74%), subpleural fat (2.62%), fractured ribs (1.24%), hilar adenopathy (0.13%), ill-defined diaphragm (0.46%), ill-defined heart border (0.29%), Kerley lines (0.06%), pleural thickening (2.35%), and tuberculosis (0.27%). The rates by age cohort for pleural abnormalities decreased significantly (30-39 years, chi2 for trend = 23.49, df = 1; 40-49 years, chi2 for trend = 176.21; 50-59 years, chi2 for trend = 401.87), but findings were not significantly different for those > or =60 years of age. This suggests that sequential age cohorts in the program are developing fewer pleural abnormalities; pleural abnormalities have historically been associated with asbestos exposure. [ABSTRACT FROM AUTHOR]

A 10-year cross-sectional analysis was conducted for 233,353 radiographic examinations performed as part of the Navy Asbestos Medical Surveillance Program. Demographic and temporal trends in abnormal radiographs were assessed during this analysis. Abnormal radiograph prevalence increased significantly with age, and abnormal radiographs were nearly 30 times more likely to occur among participants 60 to 69 years of age, compared with participants < 20 years of age (odds ratio, 27.57; 95% confidence interval, 14.75-51.53). Men were 5 times more likely than women to have an abnormal radiograph (odds ratio, 5.84; 95% confidence interval, 5.02-6.80); after controlling for differences in age, this gender association remained significant only for participants > 30 years of age. The proportion of abnormal radiographs decreased significantly over the study period [chi2 (df = 1) test for trend, chi2 = 198.7, p < 0.0001], although the cohort mean age increased. Despite aging of the Asbestos Medical Surveillance Program population, the overall prevalence of radiographic abnormalities is declining; future studies should examine the reasons for this observation. [ABSTRACT FROM AUTHOR]

This study seeks to understand the social representations (SR) of suicide among high school adolescents of a public school in Curitiba, such as the media influences on the construction of these SR. The research was performed using the qualitative method and structured around the tenets of Social Representation Theory. A total of 18 adolescents of both genders participated in the study, and data collection was conducted through the formation of focus groups. The material transcribed was analyzed by the Descending Hierarchical Classification method with the aid of the IRaMuTeQ program. It was possible to establish that the SR of the adolescents surveyed are linked to the relevance of positive interpersonal relationships when faced with the impact that suicide information causes. This predominantly defines the emotional state of the person receiving information, and the primordial hosting, listening and assistance relationships that are important for the content and manner in which situations involving suicide are reported by the media. Taking these considerations into account, new studies in this area are required to promote the development and construction of new perspectives and subsequent social empowerment through information, aiming at the enhancement of the mental health of school adolescents.

Capping ligands are vital in stabilizing various nanostructures and semiconductor quantum dots in which unusual optical properties, especially white light emission, have been realized. Oleic acid (OA) is a widely used capping ligand. Here, we report blue emission from OA in its free molecular form and further demonstrate this by anchoring OA over the surfaces of Al2O3, ZnAl2O4(ZA), ZnAl2O4:Eu3+ (ZA:Eu3+), and Y2O3:Eu3+. White light emission was observed from OA-modified ZA:Eu3+ nanophosphor due to mixing of broad blue emission of OA and red emission of Eu3+ through energy transfer from OA to Eu3+. A detailed study revealed the characteristic binding modes of OA and their dependence on Eu3+ concentration, structural inversion in ZA, and the optical properties and surface states in the pristine and OA-modified ZA:Eu3+. First principles density functional theory calculations were employed to provide an insight into the HOMO-LUMO levels of OA molecule and, electronic structure of pristine and OA-modified ZA surface. The binding of OA with the ZA:xEu3+ surface changes from bridging bidentate to chelating bidentate with increasing Eu3+ concentration in the lattice. The surface binding nature of the carboxylate group with the optimized surface of ZA and the creation of mid-gap states were deduced theoretically by using butanoic acid instead of OA. The blue emission from OA and its mixing with Eu3+ emission was further confirmed experimentally by anchoring it over Y2O3:Eu3+ red phosphor. These results show the multifunctional roles of OA as capping ligand, blue emitter and sensitizer in tuning the emission colour of red phosphors into white.

Background: Previous work indicates that 30 mg isobaric mepivacaine 1.5% plus 10 μg fentanyl produces reliable anesthesia for knee arthroscopy with a more rapid recovery profile than 45 mg mepivacaine., Questions/purposes: This randomized controlled trial compared plain mepivacaine to three reduced doses of mepivacaine with 10 μg fentanyl for spinal anesthesia., Methods: Following written informed consent, subjects undergoing outpatient knee arthroscopy were prospectively randomized into one of four groups: mepivacaine 37.5 mg (M37.5); mepivacaine 30 mg plus fentanyl 10 μg (M30/F10); mepivacaine 27 mg plus fentanyl 10 μg (M27/F10); and mepivacaine 24 mg plus fentanyl 10 μg (M24/F10). The spinal was evaluated by the blinded anesthetist and surgeon. In the post-anesthesia care unit, sensory and motor block resolution was assessed. Subjects rated their satisfaction with the overall experience., Results: Group M30/F10 (n = 6) had two "fair" anesthetics, and group M27/F10 (n = 10) had one "fair" and one "inadequate" anesthetic. Both groups were eliminated from further enrollment per study protocol. The recovery profiles showed little difference between groups M37.5 and M30/F10, except for motor block resolution (median (25th percentile, 75th percentile): 171 (135, 195) and 128 (120, 135), respectively). Groups M27/F10 and M24/F10 demonstrated recovery profiles that were faster than group M37.5. Patient satisfaction was 10/10 for all groups., Conclusions: Adding fentanyl 10 μg to a lower dose of mepivacaine 1.5% can lead to quicker recovery profiles. However, this advantage of a quicker recovery must be weighed against the likelihood of an incomplete anesthetic.

The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

Upon oxidation of the heterocyclic ring in 2'-deoxyguanosine (dG), the initial electrophilic intermediate displays a wide range of reactivities with nucleophiles leading to many downstream products. In the present study, the product profiles were mapped when aqueous solutions of dG were allowed to react with NH4Cl in the presence of the photooxidants riboflavin and Rose Bengal as well as the diffusible one-electron oxidant Na2IrCl6. Product characterization identified the 2'-deoxyribonucleosides of spiroiminodihydantoin, 5-guanidinohydantoin, and oxazolone resulting from H2O as the nucleophile. When NH3 was the nucleophile, a set of constitutional isomers that are diastereotopic were also observed, giving characteristic masses of dG + 31. ESI(+)-MS/MS of these NH3 adducts identified them to be spirocycles with substitution of either the C5 or C8 carbonyl with an amine. The NH3 adducts exhibit acid-catalyzed hydrolysis to spiroiminodihydantoin. Quantification of the NH3 and H2O adducts resulting from oxidation of dG in the nucleoside, single-stranded, and duplex oligodeoxynucleotide contexts were monitored allowing mechanisms for product formation to be proposed. These data also provide a cautionary note to those who purify their oligonucleotide samples with ammonium salts before oxidation because this will lead to unwanted side reactions in which ammonia participates in product formation.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (dOG), a well-studied oxidation product of 2'-deoxyguanosine (dG), is prone to facile further oxidation forming spiroiminodihydantoin 2'-deoxyribonucleoside (dSp) in the nucleotide pool and in single-stranded oligodeoxynucleotides (ODNs). Many methods for quantification of damaged lesions in the genome rely on digestion of DNA with exonucleases or endonucleases and dephosphorylation followed by LC-MS analysis of the resulting nucleosides. In this study, enzymatic hydrolysis of dSp-containing ODNs was investigated with snake venom phosphodiesterase (SVPD), spleen phosphodiesterase (SPD) and nuclease P1. SVPD led to formation of a dinucleotide, 5'-d(Np[Sp])-3' (N = any nucleotide) that included the undamaged nucleotide on the 5' side of dSp as the final product. This dinucleotide was a substrate for both SPD and nuclease P1. A kinetic study of the activity of SPD and nuclease P1 showed a sequence dependence on the nucleotide 5' to the lesion with rates in the order dG>dA>dT>dC. In addition, the two diastereomers of dSp underwent digestion at significantly different rates with dSp1>dSp2; nuclease P1 hydrolyzed the 5'-d(Np[Sp1])-3' dinucleotide two- to six-fold faster than the corresponding 5'-d(Np[Sp2])-3', while for SPD the difference was two-fold. These rates are chemically reasoned based on dSp diastereomer differences in the syn vs. anti glycosidic bond orientation. A method for the complete digestion of dSp-containing ODNs is also outlined based on treatment with nuclease P1 and SVPD. These findings have significant impact on the development of methods to detect dSp levels in cellular DNA.

8-Oxo-7,8-dihydroguanine (OG) is the most common base damage found in cells, where it resides in many structural contexts, including the nucleotide pool, single-stranded DNA at transcription forks and replication bubbles, and duplex DNA base-paired with either adenine (A) or cytosine (C). OG is prone to further oxidation to the highly mutagenic hydantoin products spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in a sharply pH-dependent fashion within nucleosides. In the present work, studies were conducted to determine how the structural context affects OG oxidation to the hydantoins. These studies revealed a trend in which the Sp yield was greatest in unencumbered contexts, such as nucleosides, while the Gh yield increased in oligodeoxynucleotide (ODN) contexts or at reduced pH. Oxidation of oligomers containing hydrogen-bond modulators (2,6-diaminopurine, N(4)-ethylcytidine) or alteration of the reaction conditions (pH, temperature, and salt) identify base stacking, electrostatics, and base pairing as the drivers of the key intermediate 5-hydroxy-8-oxo-7,8-dihydroguanine (5-HO-OG) partitioning along the two hydantoin pathways, allowing us to propose a mechanism for the observed base-pairing effects. Moreover, these structural effects cause an increase in the effective pK(a) of 5-HO-OG, following an increasing trend from 5.7 in nucleosides to 7.7 in a duplex bearing an OG·C base pair, which supports the context-dependent product yields. The high yield of Gh in ODNs underscores the importance of further study on this lesion. The structural context of OG also determined its relative reactivity toward oxidation, for which the OG·A base pair is ~2.5-fold more reactive than an OG·C base pair, and with the weak one-electron oxidant ferricyanide, the OG nucleoside reactivity is >6000-fold greater than that of OG·C in a duplex, leading to the conclusion that OG in the nucleoside pool should act as a protective agent for OG in the genome.

This study compared the performances of two laboratory-scale sequencing batch reactors to remove 17β-estradiol and 17α-ethinyl estradiol. Both SBRs were operated to achieve organic carbon oxidation and nitrification. However, the overall bacterial population in both SBRs was targeted to be different by feeding the SBRs with peptone and glucose. Furthermore, the reactors were also run at different solid retention times (SRTs) to evaluate the effect of SRT on estrogen removal. The more diverse heterotrophic and ammonia oxidizing bacterial community in the peptone fed SBR1 had superior estrogen removal than the glucose fed SBR 2 which enriched less diverse community, particularly for 17α-ethinyl estradiol. Under a solids retention time (SRT) of 40days, the total 17β-estradiol mass was 30% of the amount under the SRT of 20days, and the total 17α-ethinyl estradiol mass was likewise 40% of the amount under the shorter SRT., (Published by Elsevier Ltd.)

Exposure of cells to phenolic compounds through exogenous and endogenous sources can lead to deleterious effects via nucleobase modifications of DNA occurring under oxidative conditions. 2'-Deoxyguanosine (dG) is the most electron rich of the four canonical bases and includes many nucleophilic sites; it is also susceptible to oxidation with numerous reactive oxygen species. In these studies, dG was allowed to react with 2-naphthol in the presence of copper or iron salts yielding two principal isomeric products. Spectroscopic analysis and reactions with alkylated nucleosides support the assignment of compound 1a/1b as a pair of atropisomer N(2) adducts and compound 2a/2b as a diastereomeric mixture of tricyclic [4.3.3.0] adducts. Both products are the result of an overall four-electron oxidation process and consequently have the same masses, though drastically different structures, providing mechanistic insight into their formation. Thus, dG alkylation by 2-naphthol under oxidative conditions yields products whose structural properties are altered, leading to potentially mutagenic effects in genomic DNA.

Reactive oxygen species attack both base and sugar moieties in DNA with a preference among the bases for reaction at guanine. In the present study, 2'-deoxyguanosine (dG) was oxidized by a copper-mediated Fenton reaction with the reductants ascorbate or N-acetyl-cysteine, yielding oxidation on both the base and the sugar. The primary oxidized lesions observed in these studies include the 2'-deoxyribonucleosides of 8-oxo-7,8-dihydroguanosine (dOG), spiroiminodihydantoin (dSp), guanidinohydantoin (dGh), oxazolone (dZ), and 5-carboxamido-5-formamido-2-iminohydantoin (d2Ih), as well as the free base guanine. d2Ih was the major product observed in the nucleoside, single- and double-stranded oligodeoxynucleotide contexts and is proposed to arise from oxidation at C5 of guanine. Product distribution studies provide insight into the role of the reductant in partitioning of dG base oxidation along the C5 and C8 pathways.

As the most readily oxidized of DNA's four natural bases, guanine is a prime target for attack by reactive oxygen species (ROS) and transition metal-mediated oxidants. The oxidation products of a modified guanosine nucleoside and of a single-stranded oligodeoxynucleotide, 5'-d(TTTTTTTGTTTTTTT)-3' have been studied using oxidants that include Co(II), Ni(II), and Ir(IV) compounds as well as photochemically generated oxidants such as sulphate radical, electron-transfer agents (riboflavin) and singlet oxygen. The oxidized lesions formed include spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), imidazolone (Iz), oxazolone (Z) and 5-carboxamido-5-formamido-2-iminohydantion (2-Ih) nucleosides with a high degree of dependence on the exact oxidation system employed. Interestingly, a nickel(II) macrocyclic complex in conjunction with KHSO(5) leads to the recently reported 2-Ih heterocycle as the major product in both the nucleoside and oligonucleotide contexts.

Background and Objectives: This prospective, randomized, double blind study was designed to determine whether reduction in volume from 20 to 10 mL of ropivacaine 0.5% for ultrasound-guided interscalene block might decrease the incidence of diaphragmatic paresis and preserve pulmonary function., Method: Thirty patients scheduled for arthroscopic shoulder surgery were randomized to receive either 10 or 20 mL of ropivacaine 0.5% for interscalene block at the level of the cricoid cartilage. General anesthesia was administered for surgery, and the surgeon infiltrated lidocaine at the port sites. Hemidiaphragmatic excursion and pulmonary function tests were measured before block, 15 mins after block, and at the time of discharge from recovery room. Onset and duration of sensory dermatomal spread, motor block, pain scores, and analgesic consumption were recorded., Results: Hemidiaphragmatic paresis occurred 15 mins after block performance in 14 of 15 patients in each group. At postanesthesia care unit discharge, 13 of 15 patients in each group continued to demonstrate hemidiaphragmatic paresis. Significant reduction of spirometric values(forced vital capacity, forced expiratory volume at 1 sec, and peak expiratory flow) occurred to a similar degree in both groups after block.Sensory dermatomal spread, motor block, pain scores, and analgesic consumption were not significantly different between groups., Conclusions: Decreasing the volume for interscalene block from 20 to 10 mL did not reduce the incidence of hemidiaphragmatic paresis or impairment in pulmonary function, which persisted at discharge from recovery room. No significant differences in quality or duration of analgesia were observed.

Experimentally, it was observed that the oxidized guanine lesion spiroiminodihydantoin (Sp) contained in highly purified oligodeoxynucleotides slowly converts to guanidinohydantoin (Gh). The reaction is accelerated in the presence of acid. The possible mechanisms of this transformation have been analyzed computationally. Specifically, the potential energy surface for formation of Gh from Sp has been mapped using B3LYP density functional theory, the aug-cc-pVTZ and 6-31+G(d,p) basis sets, and the integral equation formalism for the polarizable continuum model (IEF-PCM) solvation model. The results favor a mechanism in which proton-assisted hydration of the C6 carbonyl group forming a gem-diol leads to ring opening of the iminohydantoin ring. The resulting species resembles a beta-ketoacid in its ability to decarboxylate; tautomerization of the resulting enol forms Gh. The results of these studies indicate that incubation of nucleosides or oligonucleotides containing Sp should be avoided in acidic media when high purity or an accurate assessment of the amounts of hydantoin lesions is desired.

8-Oxo-7,8-dihydroguanosine (8-oxoG) is among the most common forms of oxidative DNA damage found in human cells. The question of damage recognition by the repair machinery is a long standing one, and it is intriguing to suggest that the mechanism of efficiently locating damage within the entire genome might be related to modulations in the electronic properties of lesions compared to regular bases. Using laser-based methods combined with organizing various oligomers self-assembled monolayers on gold substrates, we show that indeed 8-oxoG has special electronic properties. By using oligomers containing 8-oxoG and guanine bases which were inserted in an all thymine sequences, we were able to determine the energy of the HOMO and LUMO states and the relative density of electronic states below the vacuum level. Specifically, it was found that when 8-oxoG is placed in the oligomer, the HOMO state is at higher energy than in the other oligomers studied. In contrast, the weakly mutagenic 8-oxo-7,8-dihydroadenosine (8-oxoA) has little or no effect on the electronic properties of DNA.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), a prevalent product of oxidative stress on cellular DNA, is readily further oxidized forming adducts with nucleophiles. In the presence of tyrosine or p-cresol, an unusual tricyclo[4.3.3.0] adduct has been characterized in both nucleoside and oligodeoxynucleotide studies. The adduct is more stable in oligomers than nucleosides and undergoes slow reversion and hydration to spiroiminodihydantoin.

The reaction between N(alpha)-acetyllysine methyl ester (Lys) and 2'-deoxyguanosine (dGuo) was used to study structural aspects of DNA-protein cross-link (DPC) formation. The precise structure of DPCs depended on the nature of the oxidant and cross-linking reactions in which a series of different oxidation conditions generated a distribution of adducts, principally spirodiiminodihydantoins with lysine appended at the purine position of C5 (5-Lys-Sp), C8 (8-Lys-Sp), or both C5 and C8 (5,8-diLys-Sp). Singlet oxygen oxidation of dGuo produced 5-Lys-Sp exclusively when Rose Bengal or methylene blue was used to photochemically generate (1)O2 in the presence of Lys, whereas riboflavin or benzophenone-mediated photochemistry generated lysine radicals and led to C8 adduct formation, yielding 8-Lys-Sp and 5,8-diLys-Sp. Notably, the yield of dGuo modifications from riboflavin photooxidation increased dramatically in the presence of lysine. Oxidation of deoxyguanosine/lysine mixtures with Na2IrCl6 or sulfate radicals produced both 5-Lys-Sp and 8-Lys-Sp. The same adducts were formed in single and double-stranded oligodeoxynucleotides, and these could be analyzed after nuclease digestion. Adduct formation in duplex DNA was somewhat dependent on the accessibility of lysine to C5 vs C8 of the purine. No adduct formation was detected between lysine and the other nucleobases T, C, or A. Overall, the precise location of adduct formation at C5 vs C8 of guanine appears to be diagnostic of the oxidation pathway.

Oxidation of guanine (G) and 8-oxoguanine (OG) with a wide variety of oxidants yields the hydantoin lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). These two lesions have garnered much recent attention due to their unusual structures and high mutagenic potential. We have previously shown that duplexes containing Gh and Sp are substrates for the base excision repair glycosylase Escherichia coli Fpg (EcFpg). To evaluate the recognition features of these unusual lesions, binding and footprinting experiments were performed using a glycosylase inactive variant, E3Q EcFpg, and 30 bp duplexes containing the embedded lesions. Surprisingly, E3Q EcFpg was found to bind significantly more tightly ( approximately 1000-fold) to duplexes containing Gh or Sp over the corresponding duplexes containing OG. This may be a consequence of the helix-destabilizing nature of the hydantoin lesions that facilitates their recognition within duplex DNA. Though DNA binding affinities of E3Q EcFpg with Gh- and Sp-containing duplexes were found to be similar to each other, hydroxyl radical footprinting using methidium-propyl-EDTA (MPE)-Fe(II) revealed subtle differences between binding of E3Q EcFpg to the two lesions. Most notably, in the presence of E3Q EcFpg, the Sp nucleotide (nt) is hyperreactive toward cleavage by MPE-Fe(II)-generated hydroxyl radicals, suggestive of the formation of an intercalation site for the MPE-Fe(II) reagent at the Sp nt. Interestingly, increasing the duplex length from 18 to 30 bp enhanced the excision efficiency of Gh and Sp paired with C, G, or T by EcFpg such that these substrates are processed as efficiently as the signature substrate lesion, OG. Moreover, the base removal activity with these two lesions was more efficient than removal of OG when in a base pairing context opposite A. The high affinity and efficient activity of EcFpg toward the hydantoin lesions suggest that EcFpg mediates repair of the lesions in vivo. Notably, the facile activity of EcFpg toward Gh and Sp in base pairing contexts with G and A, which are likely to be present after DNA replication, would be detrimental and enhance mutagenesis.

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.

Two convenient synthetic routes to the oxidized guanosine triphosphate lesions spiroiminodihydantoin-2'-deoxynucleoside-5'-triphosphate (dSpTP) and guanidinohydantoin-2'-deoxynucleoside-5'-triphosphate (dGhTP) are reported. Both two-electron oxidation of 2'-deoxy-7,8-dihydro-8-oxoguanosine-5'-triphosphate (dOGTP) using SO4*- generated photolytically from K2S2O8 or four-electron oxidation of 2'-deoxyguanosine-5'-triphosphate (dGTP) from singlet oxygen provide either dSpTP or dGhTP at pH 8.0 or 4.4, respectively. Highly purified triphosphates are obtained by ion pair reversed-phase HPLC.

The U.S. Navy Asbestos Medical Surveillance Program is a comprehensive effort to decrease exposure to asbestos, a known health hazard. This study was part of a programmatic review of the Asbestos Medical Surveillance Program database, which included 233,353 radiographic examinations from 1990 to 1999. The initial review focused on incidental findings recorded by B-readers for 23,460 radiographs. Abnormalities reported included bullae (0.68%), cancer (0.56%), cardiac size/ shape abnormalities (1.36%), emphysema (0.74%), subpleural fat (2.62%), fractured ribs (1.24%), hilar adenopathy (0.13%), ill-defined diaphragm (0.46%), ill-defined heart border (0.29%), Kerley lines (0.06%), pleural thickening (2.35%), and tuberculosis (0.27%). The rates by age cohort for pleural abnormalities decreased significantly (30-39 years, chi2 for trend = 23.49, df = 1; 40-49 years, chi2 for trend = 176.21; 50-59 years, chi2 for trend = 401.87), but findings were not significantly different for those > or =60 years of age. This suggests that sequential age cohorts in the program are developing fewer pleural abnormalities; pleural abnormalities have historically been associated with asbestos exposure.

The formation of covalent cross-links between amino acid side chains and DNA bases in DNA-protein complexes is a significant pathway in oxidative damage to the genome, yet much remains to be learned about their chemical structures and mechanisms of formation. In the present study, DNA-protein cross-links (DPCs) were formed between synthetic oligodeoxynucleotides containing an 8-oxo-7,8-dihydro-2'deoxyguanosine (OG) or an 8-oxo-7,8-dihydro-2'-deoxyadenosine (OA) nucleotide and Escherichia coli singled-stranded binding protein (SSB) under oxidative conditions. Studies with various sequences indicated that DNA homopolymers and those lacking 8-oxopurines were less reactive toward DPC formation. DPCs were formed in the presence of HOCl, peroxynitrite, and the one-electron oxidants Na(2)IrCl(6), Na(2)IrBr(6), and Na(3)Fe(CN)(6). Protein-protein cross-linking was also observed, particularly for oxidants of high reduction potential such as Na(2)IrCl(6). The adducted oligodeoxynucleotides were sensitive to hot piperidine treatment leading to strand scission at the site of cross-linking. In addition, the covalent cross-links were somewhat heat and acid labile, which may be related to the difficulties encountered in obtaining complete characterization of trypsin digests of the DPCs. However, model reactions involving the single amino acids lysine, arginine, and tyrosine, residues known to be involved in base contacts in the DNA:SSB complex, could be studied, and the adduct formed between N(alpha)-acetyllysine methyl ester and an 18-mer containing OG was tentatively characterized by electrospray ionization mass spectrometry as analogues of spiroiminodihydantoin and guanidinohydantoin. A mechanism involving nucleophilic attack of an amino acid side chain (e.g. the epsilon-amino group of lysine) at C5 of a 2-electron oxidized form of OG is proposed.

Oxidation of single- or double-stranded DNA containing a 7,8-dihydro-8-oxoguanosine lesion with the one-electron oxidant Na2IrCl6 in the presence of spermine led to formation of a covalent adduct that was analyzed by gel electrophoresis, HPLC, ESI-MS, and UV-vis. The adduct was labile to heat, exhibiting a t1/2 of 12 h at 37 degrees C, and the ultimate hydrolysis product was characterized as a deoxyribosylurea lesion. Data from model studies with 1,3-diaminopropane vs 1,4-diaminobutane are consistent with initial formation of a C5 spermine adduct from a dehydro-8-oxoguanosine intermediate, followed by rearrangement to a spiroaminal subject to slow hydrolysis at C4 of the purine. Spermine adducts could also be formed from oxidation of the analogous G-containing oligomer from reaction with singlet oxygen, albeit in lower yield. These results are surprising in light of the traditional view that spermine is radioprotective against DNA oxidation.

Many transition metal complexes mediate DNA oxidation in the presence of oxidizing radiation, photosensitizers, or oxidants. The final DNA oxidation products vary depending on the nature of metal complexes and the structure of DNA. Here we propose a mechanism of oxidation of a nucleotide, deoxyguanosine 5'-monophosphate (dGMP) by trans-d,l-1,2-diaminocyclohexanetetrachloroplatinum (trans-Pt(d,l)(1,2-(NH(2))(2)C(6)H(10))Cl(4), [Pt(IV)Cl(4)(dach)]; dach = diaminocyclohexane) to produce 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) stoichiometrically. The reaction was studied by high-performance liquid chromatography (HPLC), (1)H and (31)P nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). The proposed mechanism involves Pt(IV) binding to N7 of dGMP followed by cyclization via nucleophilic attack of a phosphate oxygen at C8 of dGMP. The next step is an inner-sphere, two-electron transfer to produce a cyclic phosphodiester intermediate, 8-hydroxyguanosine cyclic 5',8-(hydrogen phosphate). This intermediate slowly converts to 8-oxo-dGMP by reacting with solvent H(2)O.

Guanosine labeled with 15N at N1, amino, and N7 and 13C at either C2 or C8 was oxidized by Rose Bengal photosensitization (singlet oxygen) in buffered aqueous solution. At pH > 7, spiroiminodihydantoin was the major product, while at pH < 7, guanidinohydantoin (Gh) was the principal product. 15N and 13C NMR studies confirmed that Gh was formed as a mixture of slowly equilibrating diastereomers. Experiments conducted in H218O indicated that Gh and Sp each contained one oxygen atom derived from O2 and one from H2O. Tandem mass spectrometry was used to identify the C4 carbonyl of Gh as the one labeled with 18O, supporting a mechanism involving attack of water at C5 of a dehydro-8-oxoguanosine intermediate.

Rationale and Objectives: Electrocardiogram-gated 3D volume data from multidetector computed tomography coronary angiography (MDCT-CA) enable image reconstruction in any phase of the cardiac cycle. The objective was to determine left ventricular (LV) function parameters by MDCT in comparison to cine magnetic resonance imaging (MRI)., Method: Thirty patients with known or suspected coronary artery disease (CAD) underwent MDCT-CA. From multiplanar reformations in short axis orientation end-diastolic and end-systolic LV volumes (LVEDV, LVESV) were determined to calculate LV stroke volume and ejection fraction (LVSV, LVEF) and compared with MRI measurements., Results: LVEDV (147 +/- 27 mL) and LVESV (65 +/- 22 mL) determined by MDCT correlated well to the respective MRI measurements (LVEDV 133 +/- 27 mL, r = 0.80, P < 0.001; LVESV 48 +/- 19 mL, r = 0.89, P < 0.001). LVSV (MDCT 82 +/- 15 mL; MRI 85 +/- 17 mL; r = 0.77, P < 0.001) and LVEF (MDCT 56 +/- 9%; MRI 65 +/- 8%; r = 0.85, P < 0.001) showed a good correlation as well. LVEF was significantly underestimated by MDCT (-8.5 +/- 4.7%, P < 0.001)., Conclusions: Initial experience in patients evaluated for CAD shows that spiral MDCT studies may provide LV functional data in good correlation to Cine MRI.

8-Oxo-7,8-dihydroguanine (OG) is susceptible to further oxidation in vitro to form two secondary oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). Previous work from this laboratory has shown that OG, Gh, and Sp are recognized and excised from duplex DNA substrates by the Escherichia coli DNA repair enzyme Fpg. In this report, we extend these studies to the functionally related eukaryotic OG glycosylases (OGG) from yeast and humans: yOGG1, yOGG2, and hOGG1. The hOGG1 enzyme was active only toward the removal of 8-oxoguanine, exhibiting a 1000-fold faster rate of removal of 8-oxoguanine from OG.C-containing duplexes relative to their OG.A counterparts. Duplexes containing Gh or Sp opposite any of the four natural bases were not substrates for the hOGG1 enzyme. In contrast, both yOGG1 and yOGG2 enzymes removed Gh and Sp in a relatively efficient manner from an 18 bp duplex. No significant difference was observed in the rate of reaction of Gh- and Sp-containing duplexes with yOGG1. However, yOGG2 removed Sp at a faster rate than Gh. Both yOGG enzymes exhibit a negligible dependence on the base opposite the lesion, suggesting that the activity of these enzymes may be promutagenic. Surprisingly, in the 18 bp sequence context, both yOGG enzymes did not exhibit OG removal activity. However, both removed OG in a 30 bp duplex with a different sequence surrounding the OG. The wide range of repair efficiencies observed by these enzymes with different substrates in vitro suggests that this could greatly affect the mutagenicity of these lesions in vivo. Indeed, the greater efficiency of the yOGG proteins for removal of the further oxidized products, Gh and Sp, over their 8-oxoguanine parent, suggests that these lesions may be the preferred substrates in vivo.

Single-stranded DNA genomes have been constructed that site-specifically contain the 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG) oxidation products guanidinohydantoin (Gh) and the two stable stereoisomers of spiroiminodihydantoin (Sp1 and Sp2). The circular viral genomes were transfected into wild-type AB1157 Escherichia coli, and the efficiency of lesion bypass by DNA polymerase(s) was assessed. Viral progeny were analyzed for mutation frequency and type using the recently developed restriction endonuclease and postlabeling (REAP) assay. Gh was bypassed nearly as efficiently as the parent 8-oxoG but was highly mutagenic, causing almost exclusive G --> C transversions. The stereoisomers Sp1 and Sp2 were, in comparison, much stronger blocks to DNA polymerase extension and caused a mixture of G --> T and G --> C transversions. The ratio of G --> T to G --> C mutations for each Sp lesion was dependent on the stereochemical configuration of the base. All observed mutation frequencies were at least an order of magnitude higher than those caused by 8-oxoG. Were these lesions to be formed in vivo, our data show that they are absolutely miscoding and may be refractory to repair after translesion synthesis.

The low redox potential of 8-oxo-7,8-dihydroguanine (OG), a molecule regarded as a marker of oxidative damage in cells, makes it an easy target for further oxidation. Using a temperature-dependent method of synthesis, the oxidation products of OG, guanidinohydantoin (Gh) and/or its isomer iminoallantoin (Ia) as well as spiroiminodihydantoin (Sp), have been site-specifically incorporated into DNA oligomers. Single nucleotide insertion and primer extension experiments using Escherichia coli Kf exo(-) DNA polymerase were carried out under "standing start" and "running start" conditions in various sequence contexts. dAMP and dGMP were found to be inserted opposite these OG oxidation products. Steady-state kinetic studies show that the Gh/Ia.G base pair yields a lower K(m) value compared to the Sp.G pair or X.A (X = Gh/Ia or Sp). Running start experiments using oxidized and unoxidized OG-containing templates showed enhanced full extension in the presence of all four dNTPs. A sequence preference for efficiency of extension was found when Gh/Ia and Sp are present in the DNA template, possibly leading to primer misalignment. Full extension is more efficient for the templates containing two Gs immediately 3' to the lesions compared to two As. Although these lesions cause a significant block for DNA elongation, results show that they are more easily bypassed by the polymerase when situated in the appropriate sequence context. UV melting studies carried out on duplexes mimicking the template/primer systems were used to characterize thermal stability of the duplexes. These experiments suggest that both Gh/Ia and Sp destabilize the duplex to a much greater extent than OG, with Sp being most severe.

In vitro work in this laboratory has identified new DNA lesions resulting from further oxidation of a common biomarker of oxidative damage, 8-oxo-7,8-dihydroguanine (OG). The major product of oxidation of OG in a nucleoside, nucleotide, or single-stranded oligodeoxynucleotide using metal ions that act as one-electron oxidants is the new nucleoside derivative spiroiminodihydantoin (Sp). In duplex DNA an equilibrating mixture of two isomeric products, guanidinohydantoin (Gh) and iminoallantoin (Ia), is produced. These products are also formed by the overall four-electron oxidation of guanosine by photochemical processes involving O(2). DNA template strands containing either Sp or Gh/Ia generally acted as a block to DNA synthesis with the Klenow exo(-) fragment of pol I. However, when nucleotide insertion did occur opposite the lesions, only 2'-deoxyadenosine 5-triphosphate and 2'-deoxyguanine 5-triphosphate were used for primer extension. The Escherichia coli DNA repair enzyme Fpg was able to remove the Sp and Gh/Ia lesions from duplex DNA substrates, although the efficiency was depended on the base opposite the lesion.

This study presents mean hearing thresholds from a cross-sectional study of 68,632 monitoring audiograms submitted to the Navy Environmental Health Center for 1995 to 1999. Records included U.S. Navy and U.S. Marine Corps enlisted personnel (Navy men = 51,643; Navy women = 4,184; Marine Corps men = 12,251: Marine Corps women = 554). Mean hearing thresholds were calculated for age groups (17-24, 25-29, 30-34, 35-39, 40-44, 45-49, and 50 years and older), gender (male/female), and service (Navy/Marine Corps). Although hearing thresholds worsened with increasing age, as expected, Navy and Marine Corps men have worse levels than Occupational Safety and Health Administration age-corrected values throughout most of their careers, whereas women were closer to the Occupational Safety and Health Administration values. Hearing thresholds in the Navy have not improved appreciably from historical reports. Threshold variability, as revealed by standard deviations, increased with age and paralleled threshold levels. This epidemiological information suggests that hearing conservation continues to be an important force health protection issue.

[reaction: see text]. The riboflavin-catalyzed photooxidation of 2',3',5'-tri-O-acetyl-8-oxo-7,8-dihydroguanosine generates a radical intermediate that is competitively trapped by H(2)O, O2(-)(*), or O(2). The products of H(2)O trapping have been previously described as the spiroiminodihydantoin (pH >or= 7) and iminoallantoin/guanidinohydantoin (pH < 7) nucleosides. Trapping by O2(-)(*) leads to the oxaluric acid (pH or= 8.6) pathways (R' ', R' ' = H or 2,3,5-tri-O-Ac-ribofuranosyl). The pH-dependent role of superoxide was probed using Mn-SOD and compared to guanosine and 8-methoxyguanosine photooxidation.

Use of one-electron oxidants such as Na(2)IrCl(6) to oxidize 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) residues in oligodeoxynucleotides was previously shown to lead to predominant formation of a base lesion of mass M - 10 compared to starting material [Duarte et al. (1999) Nucleic Acids Res. 27, 596-502]. To thoroughly characterize the structure of this lesion, the oxidation of the nucleoside 9-N-(2',3',5'-tri-O-acetyl-beta-D-erythro-pentanosyl)-8-oxo-7,8-dihydroguanine with one-electron oxidants at pH 2-4 was used as a model for duplex DNA oxidation of OG residues. (1)H NMR and H,H COSY NMR studies in CD(3)OD along with LC-ESI-MS/MS fragmentation analysis are consistent with the assignment of the M - 10 species as a mixture of two pH-dependent equilibrating isomers, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) nucleoside, both present as mixtures of epimers at the C5 position of the hydantoin ring, i.e., four total isomers are formed. The Gh/Ia mixture is formed from hydration and decarboxylation of the initially formed intermediate 5-hydroxy-8-oxo-7,8-dihydroguanosine, a species that is also produced by four-electron oxidation (e.g., singlet oxygen) of guanosine. The product mixture can be further oxidized to a species designated Ia(ox), a hydrolytically unstable material at pH 7 that has been characterized by ESI-MS and (1)H NMR. Competition studies with 8-oxo-7,8-dihydroadenosine placed the redox potential of Gh/Ia at about 1.0 V vs NHE. These studies provide important information concerning the structures of lesions obtained when OG, a "hot spot" for oxidative damage, serves as a "hole trap" in long-range electron-transfer studies.

8-oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (kcat/K(m) of 0.24-0.26 min(-1) x nM(-1)), while Nei prefers G over C as the complementary base (k(cat)/K(m) - 0.15-0.17 min(-1) x nM(-1)). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG.A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp).A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh.G or Sp.G pair, but not from Gh.C and Sp.C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.

Idiopathic CD4+ T cell lymphocytopenia was unexpectedly detected in a 33-year-old, otherwise healthy young woman with no HIV or other viral infection, autoimmune, or neoplastic disease or increased susceptibility to infection. CD4+ T cell levels were 60-140/microl over a 3.5-year period. Following an uneventful pregnancy, the patient developed anemia and interstitial nephritis associated with a plasma cell dyscrasia with a monoclonal IgA gammopathy and a shifting immunoglobulin pattern that included IgG and IgA monoclonal proteins and increased urinary light chains. Osteolytic lesions were never detected and bone marrow aspirations revealed up to 10% atypical plasma cells. Various therapies often used in treating multiple myeloma only temporarily controlled the increasing renal damage. IL-2 therapy of 600,000 to 1 million units subcutaneously daily resulted in increased CD4+ T cells to normal levels, a decrease in the gammopathy, a return of renal function, energy, and weight gain, and apparently normal health status sustained for 2 years. The findings are compatible with a potentially fatal but nonmalignant immunoregulatory disorder that can be controlled by IL-2 administration., (Copyright 2001 Academic Press.)

An intriguing feature of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is that it is highly reactive toward further oxidation. Indeed, OG has been shown to be a "hot spot" for oxidative damage and susceptible to oxidation by a variety of cellular oxidants. Recent work has identified two new DNA lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), resulting from one-electron oxidation of OG. The presence of Gh and Sp lesions in DNA templates has been shown to result in misinsertion of G and A by DNA polymerases, and therefore, both are potentially mutagenic DNA lesions. The base excision repair (BER) glycosylases Fpg and MutY serve to prevent mutations associated with OG in Escherichia coli, and therefore, we have investigated the ability of these two enzymes to process DNA duplex substrates containing the further oxidized OG lesions, Gh and Sp. The Fpg protein, which removes OG and a variety of other oxidized purine base lesions, was found to remove Gh and Sp efficiently opposite all four of the natural DNA bases. The intrinsic rate of damaged base excision by Fpg was measured under single-turnover conditions and was found to be highly dependent upon the identity of the base opposite the OG, Gh, or Sp lesion; as expected, OG is removed more readily from an OG:C- than an OG:A-containing substrate. However, when adenine is paired with Gh or Sp, the rate of removal of these damaged lesions by Fpg was significantly increased relative to the rate of removal of OG from an OG:A mismatch. The adenine glycosylase MutY, which removes misincorporated A residues from OG:A mismatches, is unable to remove A paired with Gh or Sp. Thus, the activity of Fpg on Gh and Sp lesions may dramatically influence their mutagenic potential. This work suggests that, in addition to OG, oxidative products resulting from further oxidation of OG should be considered when evaluating oxidative DNA damage and its associated effects on DNA mutagenesis.

[reaction: see text] Further oxidation of the common DNA lesion 8-oxo-7,8-dihydroguanosine by one-electron oxidants such as IrCl6(2-), Fe(CN)6(3-), or SO4-* leads to two major products, depending upon reaction conditions. In nucleosides at pH 7, 22 degrees C, the principal product is shown herein to be a spiroiminodihydantoin nucleoside, as a diastereomeric mixture, that can be characterized by NMR, ESI-MS/MS, and independent synthesis.

The structural characteristics of Z-DNA were used to challenge the selectivity of guanine oxidation promoted by nickel and cobalt reagents. Base pairing and stacking within all helical structures studied previously had hindered access to guanine and limited its reaction. However, the Z-helix uniquely retains high exposure of guanine N7. This exposure was sufficient to direct oxidation specifically to a plasmid insert -(CG)(13)AATT(CG)(13)- that adopted a Z-conformation under native supercoiling. An alternative insert -(CG)(7)- retained its B-conformation and demonstrated the expected lack of reactivity. For a nickel salen complex made from a particularly bulky ligand, preferential reaction shifted to the junctions within the Z-DNA insert as is common for large reagents. Inactivation of the nickel reagents by high-salt concentrations prevented parallel investigations of Z-DNA, formed by oligonucleotides. However, the activity of Co(2+) was minimally affected by salt and consequently confirmed the high reactivity of 5'-p(CG)(4) in its Z-conformation. These reagents may now be applied to a broad array of targets, since their structural specificity remains predictable for both complex and helical assemblies of nucleic acids.

The reactions of the water-soluble complexes [NiCR](2+) (where CR = 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaene) and [NiKGH-CONH(2)](+) (where KGH-CONH(2) = lysylglycylhistidinecarboxamide) with sulfite/O(2) and peroxymonosulfate have been investigated using spectrophotometric and rapid-scan techniques. In most cases, the spectral changes suggest the formation of an intermediate Ni(III) species, followed by decomposition reactions which involve a back-reaction to Ni(II). Only in the case of the [NiCR](2+)-S(IV)-O(2) system is the formed Ni(III) species stable in solution. When sulfite and oxygen are used to oxidize Ni(II) to Ni(III), the reaction is oxygen dependent and an induction period could be observed, whereas the use of the strong oxidizing agent peroxymonosulfate resulted in no induction period and no oxygen dependence. In addition, the oxidation of Ni(II) to Ni(III) was faster if peroxymonosulfate was used instead of sulfite/O(2). The [NiKGH-CONH(2)](+) complex reacts much faster with sulfite/O(2) and peroxymonosulfate than the [NiCR](2+) does. Rate constants for the oxidation process and possible reaction mechanisms, based on available literature data, that can account for the observed kinetic observations in a qualitative way are presented, and the results are correlated with previously obtained data on DNA modification using these systems.

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.

Two gel electrophoretic methods are described for detection of 7, 8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine based on their further oxidation with one-electron oxidants including IrCl62-and IrBr62-. The products of nucleobase oxidation lead to enhanced piperidine-sensitive cleavage and to highly visible stop points in a primer extension assay. 8-oxoG and 8-oxoA lesions may be distinguished by the latter's inability to be oxidized by IrBr62-compared to IrCl62-Comparison is also made to oxidation by MnO4-.

Nickel(II) complexes of tetradentate ligands such as cyclam and salen are catalysts for olefin epoxidation using PhIO and NaOCl, respectively. In order to understand the lack of enantioselectivity observed with chiral cyclam and salen complexes, studies of DNA and RNA oxidation were carried out in which evidence for diffusible oxidants might be found. A variety of square-planar, tetradentate nickel(II) complexes were observed to mediate guanine-specific modification in the presence of KHSO5 or magnesium monoperphthalate. In particular, the cationic complex, [(2,12-dimethyl-3,7,11,17-tetraazabicyclo [11.3.1]heptadeca-1(17),2,11,13,15-pentaenato)nickel]2+, [NiCR]2+, has been studied as a probe of nucleic acid folding. The extent of guanine reaction depends upon the exposure of N7, a good transition metal binding site, thus implicating nickel-guanine binding during DNA oxidation. If this is the case, related systems should be able to confer enantioselectivity during the use of chiral nickel complexes and achiral substrates for oxidation. Mechanistic studies, including radical quenching and DNA enantioselectivity, are described and their mechanistic implications discussed.

Seven new steroidal polyamines derived from bile acids, either lithocholic or deoxycholic acid, have been studied as DNA-binding agents using four complimentary methods: an ethidium displacement assay, observed changes in the thermal denaturation of poly[d(AT)], effects on hyperchromicity of DNA, and circular dichroism. In addition, modelling studies were conducted to examine the electrostatic surface potential of the polycations. The results point to a key role for a large hydrophobic surface area on the steroid in addition to the Coulombic attraction by ammonium and guanidinium groups on the steroid interacting with the polyphosphate backbone.

Seven new amino- and guanidino-substituted steroids have been synthesized from bile acid precursors, either deoxycholic acid or lithocholic acid. Their DNA binding properties have been examined using an ethidium displacement assay, through studies of hyperchromicity and thermal denaturation, and by circular dichroism. Comparison is made to simple aliphatic polyamines such as putrescine, 1,12-diaminododecane, spermidine, and spermine.