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Sample records for bulky adducts final

  1. Base sequence effects on DNA replication influenced by bulky adducts. Final report, March 1, 1995--February 28, 1997

    SciTech Connect

    Geacintov, N.E.

    1997-05-31

    Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that are present in air, food, and water. While PAH compounds are chemically inert and are sparingly soluble in aqueous solutions, in living cells they are metabolized to a variety of oxygenated derivatives, including the high mutagenic and tumorigenic diol epoxide derivatives. The diol epoxides of the sterically hindered fjord region compound benzo[c]phenanthrene (B[c]PhDE) are among the most powerful tumorigenic compounds in animal model test systems. In this project, site-specifically modified oligonucleotides containing single B[c]PhDE-N{sup 6}-dA lesions derived from the reactions of the 1S,2R,3R,4S and 1R,2S,3S,4R diol epoxides of B[c]PhDE with dA residues were synthesized. The replication of DNA catalyzed by a prokaryotic DNA polymerase (the exonuclease-free Klenow fragment E. Coli Po1 I) in the vicinity of the lesion at base-specific sites on B[c]PhDE-modified template strands was investigated in detail. The Michaelis-Menten parameters for the insertion of single deoxynucleotide triphosphates into growing DNA (primer) strands using the modified dA* and the bases just before and after the dA* residue as templates, depend markedly on the stereochemistry of the B[c]PhDE-modified dA residues. These observations provide novel insights into the mechanisms by which bulky PAH-DNA adducts affect normal DNA replication.

  2. Bulky DNA adducts in white blood cells: a pooled analysis of 3600 subjects

    PubMed Central

    Ricceri, Fulvio; Godschalk, Roger; Peluso, Marco; Phillips, David H.; Agudo, Antonio; Georgiadis, Panos; Loft, Steffen; Tjonneland, Anne; Raaschou-Nielsen, Ole; Palli, Domenico; Perera, Frederica; Vermeulen, Roel; Taioli, Emanuela; Sram, Radim J.; Munnia, Armelle; Rosa, Fabio; Allione, Alessandra; Matullo, Giuseppe; Vineis, Paolo

    2013-01-01

    Background Bulky DNA adducts are markers of exposure to genotoxic aromatic compounds, which reflect an individual’s ability to metabolically activate carcinogens and to repair DNA damage. Polycyclic aromatic hydrocarbons (PAH) represent a major class of carcinogens that are capable of forming such adducts. Factors that have been reported to be related to DNA adduct levels include smoking, diet, body mass index (BMI), genetic polymorphisms, the season of collection of biologic material, and air pollutants. Methods We pooled eleven studies (3,600 subjects) in which bulky DNA adducts were measured in human white blood cells with similar 32P-postlabelling techniques and for which a similar set of variables was available, including individual data on age, gender, ethnicity, batch, smoking habits, BMI, season of blood collection and a limited set of gene variants. Results Lowest DNA adduct levels were observed in the spring (median 0.50 adducts per 108 nucleotides), followed by summer (0.64), autumn (0.70) and winter (0.85) (p=0.006). The same pattern emerged in multivariate analysis, but only among never smokers (p=0.02). Adduct levels were significantly lower (p=0.001) in Northern Europe (the Netherlands, Denmark) (mean 0.60, median 0.40) than in Southern Europe (Italy, Spain, France, Greece) (mean 0.79, median 0.60). Conclusions In this large pooled analysis, we have found only weak associations between bulky DNA adducts and exposure variables. Seasonality (with higher adducts levels in winter) and air pollution may partly explain some of the inter-area differences (North vs South Europe), but most inter-area and inter-individual variation in adduct levels still remain unexplained. Impact Our study describes the largest pooled analysis of bulky DNA adducts so far, showing that inter-individual variation is still largely unexplained, though seasonality appears to play a role. PMID:20921335

  3. PURIFICATION AND RECOVERY OF BULKY HYDROPHOBIC DNA ADDUCTS

    EPA Science Inventory

    For many years 32P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of ma...

  4. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    DOE PAGES

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N -nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O 6more » -alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects.« less

  5. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    PubMed Central

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N-nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O6-alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects. PMID:21234336

  6. Xenobiotic metabolism induction and bulky DNA adducts generated by particulate matter pollution in BEAS-2B cell line: geographical and seasonal influence.

    PubMed

    Lepers, Capucine; André, Véronique; Dergham, Mona; Billet, Sylvain; Verdin, Anthony; Garçon, Guillaume; Dewaele, Dorothée; Cazier, Fabrice; Sichel, François; Shirali, Pirouz

    2014-06-01

    Airborne particulate matter (PM) toxicity is of growing interest as diesel exhaust particles have been classified as carcinogenic to humans. However, PM is a mixture of chemicals, and respective contribution of organic and inorganic fractions to PM toxicity remains unclear. Thus, we analysed the link between chemical composition of PM samples and bulky DNA adduct formation supported by CYP1A1 and 1B1 genes induction and catalytic activities. We used six native PM samples, collected in industrial, rural or urban areas, either during the summer or winter, and carried out our experiments on the human bronchial epithelial cell line BEAS-2B. Cell exposure to PM resulted in CYP1A1 and CYP1B1 genes induction. This was followed by an increase in EROD activity, leading to bulky DNA adduct formation in exposed cells. Bulky DNA adduct intensity was associated to global EROD activity, but this activity was poorly correlated with CYPs mRNA levels. However, EROD activity was correlated with both metal and polycyclic aromatic hydrocarbon (PAH) content. Finally, principal components analysis revealed three clusters for PM chemicals, and suggested synergistic effects of metals and PAHs on bulky DNA adduct levels. This study showed the ability of PM samples from various origins to generate bulky DNA adducts in BEAS-2B cells. This formation was promoted by increased expression and activity of CYPs involved in PAHs activation into reactive metabolites. However, our data highlight that bulky DNA adduct formation is only partly explained by PM content in PAHs, and suggest that inorganic compounds, such as iron, may promote bulky DNA adduct formation by supporting CYP activity.

  7. Maternal diet and dioxin-like activity, bulky DNA adducts and micronuclei in mother-newborns.

    PubMed

    Pedersen, Marie; Halldorsson, Thorhallur I; Autrup, Herman; Brouwer, Abraham; Besselink, Harrie; Loft, Steffen; Knudsen, Lisbeth E

    2012-06-01

    Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006-2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet was estimated on the basis of maternal food frequency questionnaire (FFQ) completed by the end of pregnancy. Biomarkers were detected in paired blood samples through the dioxin-responsive chemical-activated luciferase expression (CALUX)(®) bioassay, (32)P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal (β 95%CI; 0.46 (0.08, 0.84)) and cord blood (β 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size.

  8. Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.

    PubMed

    Ruan, Q; Zhuang, P; Li, S; Perlow, R; Srinivasan, A R; Lu, X J; Broyde, S; Olson, W K; Geacintov, N E

    2001-09-04

    The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus

  9. Exposure to meat-derived carcinogens and bulky DNA adduct levels in normal-appearing colon mucosa.

    PubMed

    Ho, Vikki; Brunetti, Vanessa; Peacock, Sarah; Massey, Thomas E; Godschalk, Roger W L; van Schooten, Frederik J; Ashbury, Janet E; Vanner, Stephen J; King, Will D

    2017-09-01

    Meat consumption is a risk factor for colorectal cancer. This research investigated the relationship between meat-derived carcinogen exposure and bulky DNA adduct levels, a biomarker of DNA damage, in colon mucosa. Least squares regression was used to examine the relationship between meat-derived carcinogen exposure (PhIP and meat mutagenicity) and bulky DNA adduct levels in normal-appearing colon tissue measured using (32)P-postlabelling among 202 patients undergoing a screening colonoscopy. Gene-diet interactions between carcinogen exposure and genetic factors relevant to biotransformation and DNA repair were also examined. Genotyping was conducting using the MassARRAY(®) iPLEX(®) Gold SNP Genotyping assay. PhIP and higher meat mutagenicity exposures were not associated with levels of bulky DNA adducts in colon mucosa. The XPC polymorphism (rs2228001) was found to associate with bulky DNA adduct levels, whereby genotypes conferring lower DNA repair activity were associated with higher DNA adduct levels than the normal activity genotype. Among individuals with genotypes associated with lower DNA repair (XPD, rs13181 and rs1799179) or detoxification activity (GSTP1, rs1695), higher PhIP or meat mutagenicity exposures were associated with higher DNA adduct levels. Significant interactions between the XPC polymorphism (rs2228000) and both dietary PhIP and meat mutagenicity on DNA adduct levels was observed, but associations were inconsistent with the a priori hypothesized direction of effect. Exposure to meat-derived carcinogens may be associated with increased DNA damage occurring directly in the colon among genetically susceptible individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.

    PubMed

    Kemp, Michael G; Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

  11. DNA bulky adducts in a Mediterranean population correlate with environmental ozone concentration, an indicator of photochemical smog.

    PubMed

    Palli, Domenico; Saieva, Calogero; Grechi, Daniele; Masala, Giovanna; Zanna, Ines; Barbaro, Antongiulio; Decarli, Adriano; Munnia, Armelle; Peluso, Marco

    2004-03-01

    Ozone (O(3)), the major oxidant component in photochemical smog, mostly derives from photolysis of nitrogen dioxide. O(3) may have biologic effects directly and/or via free radicals reacting with other primary pollutants and has been reported to influence daily mortality and to increase lung cancer risk. Although DNA damage may be caused by ozone itself, only other photochemical reaction products (as oxidised polycyclic aromatic hydrocarbons) may form bulky DNA adducts, a reliable biomarker of genotoxic damage and cancer risk, showing a seasonal trend. In a large series consisting of 320 residents in the metropolitan area of Florence, Italy, enrolled in a prospective study for the period 1993-1998 (206 randomly sampled volunteers, 114 traffic-exposed workers), we investigated the correlation between individual levels of DNA bulky adducts and a cumulative O(3) exposure score. The average O(3) concentrations were calculated for different time windows (0-5 to 0-90 days) prior to blood drawing for each participant, based on daily measurements provided by the local monitoring system. Significant correlations between DNA adduct levels and O3 cumulative exposure scores in the last 2-8 weeks before enrollment emerged in never smokers. Correlations were highest in the subgroup of never smokers residing in the urban area and not occupationally exposed to vehicle traffic pollution, with peak values for average concentrations 4-6 weeks before enrollment (r = 0.34). Our current findings indicate that DNA adduct formation may be modulated by individual characteristics and by the cumulative exposure to environmental levels of ozone in the last 4-6 weeks, possibly through ozone-associated reactive pollutants.

  12. Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures.

    PubMed

    de Kok, T M C M; Moonen, H J J; van Delft, J; van Schooten, F J

    2002-10-05

    It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines. Copyright 2002 Elsevier Science B.V.

  13. Analysis of biomarkers in a Czech population exposed to heavy air pollution. Part I: bulky DNA adducts.

    PubMed

    Rossner, Pavel; Svecova, Vlasta; Schmuczerova, Jana; Milcova, Alena; Tabashidze, Nana; Topinka, Jan; Pastorkova, Anna; Sram, Radim J

    2013-01-01

    The health of human populations living in industrial regions is negatively affected by exposure to environmental air pollutants. In this study, we investigated the impact of air pollution on a cohort of subjects living in Ostrava, a heavily polluted industrial region and compared it with a cohort of individuals from the relatively clean capital city of Prague. This study consisted of three sampling periods differing in the concentrations of major air pollutants (winter 2009, summer 2009 and winter 2010). During all sampling periods, the study subjects from Ostrava region were exposed to significantly higher concentrations of benzo[a]pyrene (B[a]P) and benzene than the subjects in Prague as measured by personal monitors. Pollution by B[a]P, particulate matter of aerodynamic diameter <2.5 µm (PM2.5) and benzene in the Ostrava region measured by stationary monitors was also higher than in Prague, with the exception of PM2.5 in summer 2009 when concentration of the pollutant was significantly elevated in Prague. To evaluate DNA damage in subjects from both locations we determined the levels of bulky DNA adducts in peripheral blood lymphocytes using the (32)P-postlabeling method. Despite higher B[a]P air pollution in the Ostrava region during all sampling periods, the levels of B[a]P-like DNA adducts per 10(8) nucleotides were significantly higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10(8) nucleotides, P < 0.001 for Prague and Ostrava subjects, respectively; P < 0.001). During the other two sampling periods, the levels of B[a]P-like DNA adducts were significantly higher in the Prague subjects (P < 0.001). Multivariate analyses conducted among subjects from Ostrava and Prague separately during all sampling periods revealed that exposure to B[a]P and PM2.5 significantly increased levels of B[a]P-like DNA adducts in the Ostrava subjects, but not in subjects from Prague.

  14. Environmental, Dietary, Maternal, and Fetal Predictors of Bulky DNA Adducts in Cord Blood: A European Mother–Child Study (NewGeneris)

    PubMed Central

    Mendez, Michelle A.; Schoket, Bernadette; Godschalk, Roger W.; Espinosa, Ana; Landström, Anette; Villanueva, Cristina M.; Merlo, Domenico F.; Fthenou, Eleni; Gracia-Lavedan, Esther; van Schooten, Frederik-J.; Hoek, Gerard; Brunborg, Gunnar; Meltzer, Helle M.; Alexander, Jan; Nielsen, Jeanette K.; Sunyer, Jordi; Wright, John; Kovács, Katalin; de Hoogh, Kees; Gutzkow, Kristine B.; Hardie, Laura J.; Chatzi, Leda; Knudsen, Lisbeth E.; Anna, Lívia; Ketzel, Matthias; Haugen, Margaretha; Botsivali, Maria; Nieuwenhuijsen, Mark J.; Cirach, Marta; Toledano, Mireille B.; Smith, Rachel B.; Fleming, Sarah; Agramunt, Silvia; Kyrtopoulos, Soterios A.; Lukács, Viktória; Kleinjans, Jos C.; Segerbäck, Dan; Kogevinas, Manolis

    2015-01-01

    Background: Bulky DNA adducts reflect genotoxic exposures, have been associated with lower birth weight, and may predict cancer risk. Objective: We selected factors known or hypothesized to affect in utero adduct formation and repair and examined their associations with adduct levels in neonates. Methods: Pregnant women from Greece, Spain, England, Denmark, and Norway were recruited in 2006–2010. Cord blood bulky DNA adduct levels were measured by the 32P-postlabeling technique (n = 511). Diet and maternal characteristics were assessed via questionnaires. Modeled exposures to air pollutants and drinking-water disinfection by-products, mainly trihalomethanes (THMs), were available for a large proportion of the study population. Results: Greek and Spanish neonates had higher adduct levels than the northern European neonates [median, 12.1 (n = 179) vs. 6.8 (n = 332) adducts per 108 nucleotides, p < 0.001]. Residence in southern European countries, higher maternal body mass index, delivery by cesarean section, male infant sex, low maternal intake of fruits rich in vitamin C, high intake of dairy products, and low adherence to healthy diet score were statistically significantly associated with higher adduct levels in adjusted models. Exposure to fine particulate matter and nitrogen dioxide was associated with significantly higher adducts in the Danish subsample only. Overall, the pooled results for THMs in water show no evidence of association with adduct levels; however, there are country-specific differences in results with a suggestion of an association in England. Conclusion: These findings suggest that a combination of factors, including unknown country-specific factors, influence the bulky DNA adduct levels in neonates. Citation: Pedersen M, Mendez MA, Schoket B, Godschalk RW, Espinosa A, Landström A, Villanueva CM, Merlo DF, Fthenou E, Gracia-Lavedan E, van Schooten FJ, Hoek G, Brunborg G, Meltzer HM, Alexander J, Nielsen JK, Sunyer J, Wright J, Kovács K, de

  15. Bulky DNA Adducts in Cord Blood, Maternal Fruit-and-Vegetable Consumption, and Birth Weight in a European Mother–Child Study (NewGeneris)

    PubMed Central

    Schoket, Bernadette; Godschalk, Roger W.; Wright, John; von Stedingk, Hans; Törnqvist, Margareta; Sunyer, Jordi; Nielsen, Jeanette K.; Merlo, Domenico F.; Mendez, Michelle A.; Meltzer, Helle M.; Lukács, Viktória; Landström, Anette; Kyrtopoulos, Soterios A.; Kovács, Katalin; Knudsen, Lisbeth E.; Haugen, Margaretha; Hardie, Laura J.; Gützkow, Kristine B.; Fleming, Sarah; Fthenou, Eleni; Farmer, Peter B.; Espinosa, Aina; Chatzi, Leda; Brunborg, Gunnar; Brady, Nigel J.; Botsivali, Maria; Arab, Khelifa; Anna, Lívia; Alexander, Jan; Agramunt, Silvia; Kleinjans, Jos C.; Segerbäck, Dan; Kogevinas, Manolis

    2013-01-01

    Background: Tobacco-smoke, airborne, and dietary exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with reduced prenatal growth. Evidence from biomarker-based studies of low-exposed populations is limited. Bulky DNA adducts in cord blood reflect the prenatal effective dose to several genotoxic agents including PAHs. Objectives: We estimated the association between bulky DNA adduct levels and birth weight in a multicenter study and examined modification of this association by maternal intake of fruits and vegetables during pregnancy. Methods: Pregnant women from Denmark, England, Greece, Norway, and Spain were recruited in 2006–2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires. Results: Adduct levels in maternal and cord blood samples were similar and positively correlated (median, 12.1 vs. 11.4 adducts in 108 nucleotides; Spearman rank correlation coefficient = 0.66, p < 0.001). Cord blood adduct levels were negatively associated with birth weight, with an estimated difference in mean birth weight of –129 g (95% CI: –233, –25 g) for infants in the highest versus lowest tertile of adducts. The negative association with birth weight was limited to births in Norway, Denmark, and England, the countries with the lowest adduct levels, and was more pronounced in births to mothers with low intake of fruits and vegetables (–248 g; 95% CI: –405, –92 g) compared with those with high intake (–58 g; 95% CI: –206, 90 g) Conclusions: Maternal exposure to genotoxic agents that induce the formation of bulky DNA adducts may affect intrauterine growth. Maternal fruit and vegetable consumption may be protective. Citation: Pedersen M, Schoket B, Godschalk RW, Wright J, von Stedingk H, Törnqvist M, Sunyer J, Nielsen JK, Merlo DF, Mendez MA, Meltzer HM, Lukács V, Landström A, Kyrtopoulos SA, Kovács K, Knudsen LE, Haugen

  16. Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers.

    PubMed

    Denissenko, M F; Pao, A; Pfeifer, G P; Tang, M

    1998-03-12

    Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

  17. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells.

    PubMed

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD>KO>WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N'-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA. Copyright © 2016 Elsevier

  18. Sex differences in risk of lung cancer: Expression of genes in the PAH bioactivation pathway in relation to smoking and bulky DNA adducts.

    PubMed

    Mollerup, Steen; Berge, Gisle; Baera, Rita; Skaug, Vidar; Hewer, Alan; Phillips, David H; Stangeland, Lodve; Haugen, Aage

    2006-08-15

    It is controversial whether women have a higher lung cancer susceptibility compared to men. We previously reported higher levels of smoking-related bulky DNA adducts in female lungs. In a pilot study (27 cases), we also found a higher level of female lung cytochrome P4501A1 (CYP1A1) gene expression. In the present extended study we report on the pulmonary expression of several genes involved in polycyclic aromatic hydrocarbon bioactivation in relation to sex, smoking and DNA adducts. CYP1A1, CYP1B1, aryl hydrocarbon receptor and microsomal epoxide hydrolase gene expression was measured by quantitative real-time reverse transcriptase-PCR in 121 normal lung tissue samples. The expression of CYP1A1 and CYP1B1 was significantly higher among current smokers compared to ex-smokers and never-smokers. Among current smokers, females had a 3.9-fold higher median level of CYP1A1 compared to males (p = 0.011). CYP1B1 expression was not related to sex. Lung DNA adducts (measured by 32P-postlabeling) were highly significantly related to CYP1A1 (p < 0.0001) irrespective of smoking-status. Our results are consistent with the hypothesis that CYP1A1 plays a significant role in lung DNA adduct formation and support a higher susceptibility to lung cancer among females. Copyright 2006 Wiley-Liss, Inc

  19. Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models.

    PubMed

    Westberg, Emelie; Hedebrant, Ulla; Haglund, Johanna; Alsberg, Tomas; Eriksson, Johan; Seidel, Albrecht; Törnqvist, Margareta

    2014-02-01

    Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.

  20. Malondialdehyde–deoxyguanosine and bulky DNA adducts in schoolchildren resident in the proximity of the Sarroch industrial estate on Sardinia Island, Italy

    PubMed Central

    Peluso, Marco

    2013-01-01

    Air quality is a primary environmental concern in highly industrialised areas, with potential health effects in children residing nearby. The Sarroch industrial estate in Cagliari province, Sardinia Island, Italy, hosts the world’s largest power plant and the second largest European oil refinery and petrochemical park. This industrial estate produces a complex mixture of air pollutants, including benzene, heavy metals and polycyclic aromatic hydrocarbons. Thus, we conducted a cross-sectional study to evaluate the prevalence of malondialdehyde–deoxyguanosine adducts in the nasal epithelium of 75 representative children, aged 6–14 years, attending primary and secondary schools in Sarroch in comparison with 73 rural controls. Additionally, the levels of bulky DNA adducts were analysed in a subset of 62 study children. DNA damage was measured by 32P-postlabelling methodologies. The air concentrations of benzene and ethyl benzene were measured in the school gardens of Sarroch and a rural village by diffusive samplers. Outdoor measurements were also performed in other Sarroch areas and in the proximity of the industrial estate. The outdoor levels of benzene and ethyl benzene were significantly higher in the school gardens of Sarroch than in the rural village. Higher concentrations were also found in other Sarroch areas and in the vicinity of the industrial park. The mean levels of malondialdehyde–deoxyguanosine adducts per 108 normal nucleotides ± standard error (SE) were 74.6±9.1 and 34.1±4.4 in the children from Sarroch and the rural village, respectively. The mean ratio was 2.53, 95% confidence interval (CI): 1.71–2.89, P < 0.001, versus rural controls. Similarly, the levels of bulky DNA adducts per 108 normal nucleotides ± SE were 2.9±0.4 and 1.6±0.2 in the schoolchildren from Sarroch and the rural village, respectively. The means ratio was 1.90, 95% CI: 1.25–2.89, P = 0.003 versus rural controls. Our study indicates that children residing near the

  1. Immunofluorescent detection of 8-oxo-dG and PAH bulky adducts in fish liver and mussel digestive gland.

    PubMed

    Machella, Nicola; Regoli, Francesco; Santella, Regina M

    2005-03-04

    Development of methodologies to detect DNA damage induced by pollutants is of increasing concern in marine ecotoxicology. We previously described an immunoperoxidase method for revealing 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) in marine organisms. In this work, the approach was extended to immunofluorescence detection and to the use of another antibody for polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Specimens of European eel (Anguilla anguilla) and Mediterranean mussel (Mytilus galloprovincialis) were exposed to benzo(a)pyrene (B[a]P), as model chemical carcinogen to induce both oxidized bases and B[a]P diol-epoxide-deoxyguanosine adducts. Cryostat sections of liver and digestive gland from both species were immunostained, and DNA damage was semiquantitatively evaluated by an image analysis system. Compared to untreated organisms, B[a]P-exposed organisms exhibited increased levels of oxidative DNA damage; in eels, which rapidly metabolize PAHs, the occurrence of B[a]P-DNA adducts was also detected. The immunofluorescent assay maintained all the advantages previously reported for the immunoperoxidase protocol. Both methods were tested on the same eel specimens and the immunofluorescence method showed a greater extent of relative DNA damage and a higher sensitivity. Although field validation is being carried out, our results indicate the utility of antibodies to rapidly detect DNA alterations in aquatic organisms, and to investigate the risk associated with genotoxins in marine environment.

  2. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

    PubMed Central

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide

  3. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

    PubMed

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Jia, Lei; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue-DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision

  4. Chromium (VI) induces both bulky DNA adducts and oxidative DNA damage at adenines and guanines in the p53 gene of human lung cells

    PubMed Central

    Arakawa, Hirohumi; Weng, Mao-wen; Tang, Moon-shong

    2012-01-01

    Chromium (VI) [Cr(VI)], a ubiquitous environmental carcinogen, is generally believed to induce mainly mutagenic binary and ternary Cr(III)–deoxyguanosine (dG)-DNA adducts in human cells. However, both adenine (A) and guanine (G) mutations are found in the p53 gene in Cr exposure-related lung cancer. Using UvrABC nuclease and formamidopyrimidine glycosylase (Fpg), and ligation-mediated PCR methods, we mapped the distribution of bulky DNA adducts (BDA) and oxidative DNA damage (ODD) in the p53 gene in Cr(VI)-treated human lung cells. We found that both BDA and ODD formed at 2ʹ-deoxyadenosine (dA) and dG bases. To understand the causes for these Cr-induced DNA damages, we mapped the distribution of BDA adducts and ODD in the p53 gene DNA fragments induced by Cr(III), Cr(VI) and Cr(V), the three major cellular Cr forms. We found that (i) dA at –CA- is a major Cr(VI) binding site followed by -GG- and –G-. Cr(VI) does not bind to –GGG-, (ii) Cr(VI)–DNA binding specificity is distinctly different from the Cr(III)–DNA binding in which –GGG- and –GG- are preferential sites, (iii) Cr(V) binding sites include all of Cr(VI) and Cr(III)–DNA binding sites and (iv) Cr(VI) and Cr(V) induce Fpg-sensitive sites at –G-. Together, these results suggest that Cr(VI) induction of BDA and ODD at dA and dG residues is through Cr(V) intermediate. We propose that these Cr(VI)-induced BDA and ODD contribute to mutagenesis of the p53 gene that leads to lung carcinogenesis. PMID:22791815

  5. Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

    PubMed Central

    Vyas, Rajan; Efthimiopoulos, Georgia; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2015-01-01

    1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism. PMID:26327169

  6. Interactions between CYP1A1 polymorphisms and exposure to environmental tobacco smoke in the modulation of lymphocyte bulky DNA adducts and chromosomal aberrations.

    PubMed

    Georgiadis, Panagiotis; Topinka, Jan; Vlachodimitropoulos, Dimitris; Stoikidou, Melpomeni; Gioka, Maria; Stephanou, Georgia; Autrup, Herman; Demopoulos, Nikolaos A; Katsouyanni, Klea; Sram, Radim; Kyrtopoulos, Soterios A

    2005-01-01

    CYP1A1 plays an important role in the metabolic activation of polycyclic aromatic hydrocarbons (PAH), carcinogenic components of air pollution. The influence of CYP1A1 genotype (*2A, *2B and *4) on the levels of lymphocyte bulky DNA adducts and the frequency of cells with aberrant chromosomes was assessed in 194 non-smoking subjects in whom recent exposure to environmental tobacco smoke (ETS) and airborne particulate-associated PAH were measured during two consecutive seasons (winter and summer). While CYP1A1*4 had no consistent effect on either biomarker of genetic damage, the levels of both biomarkers responded in a parallel fashion to changes in exposure/CYP1A1*2A genotype combinations during both seasons. Specifically, the levels of both biomarkers were increased in carriers of at least one CYP1A1*2A allele, as compared with CYP1A1*1 homozygotes, in subjects with ETS exposures >0.8 h/day during the previous 4 days and mean personal exposure to benzo[a]pyrene <0.9 ng/m3 during the previous 24 h (all P < 0.05). Outside these exposure limits the differential effect in CYP1A1*2A variants was lost. Although the numbers of subjects with the CYP1A1*2B polymorphism was small, the same trend appeared to be followed in this case. These effects are interpreted as resulting from differential induction of CYP1A1 expression in CYP1A1*2A and CYP1A1*2A/*2B carriers by components of ETS-polluted air at levels of exposure readily suffered by large segments of the general population and suggest that subjects with these genotypes may have increased susceptibility to the genotoxic effects of ETS.

  7. Versatility of Y-family Sulfolobus solfataricus DNA Polymerase Dpo4 in Translesion Synthesis Past Bulky N[superscript 2]-Alkylguanine Adducts

    SciTech Connect

    Zhang, Huidong; Eoff, Robert L.; Kozekov, Ivan D.; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter

    2009-09-25

    In contrast to replicative DNA polymerases, Sulfolobus solfataricus Dpo4 showed a limited decrease in catalytic efficiency (k{sub cat}/K{sub m}) for insertion of dCTP opposite a series of N{sup 2}-alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity was maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increased slightly going from the N{sup 2}-NaphG to the N{sup 2}-AnthG substrate, at the cost of fidelity. Pre-steady-state kinetic bursts were observed for dCTP incorporation throughout the series (N{sup 2}-MeG to N{sup 2}-AnthG), with a decrease in the burst amplitude and k{sub pol}, the rate of single-turnover incorporation. The pre-steady-state kinetic courses with G and all of the six N{sup 2}-alkyl G adducts could be fit to a general DNA polymerase scheme to which was added an inactive complex in equilibrium with the active ternary Dpo4 {center_dot} DNA {center_dot} dNTP complex, and only the rates of equilibrium with the inactive complex and phosphodiester bond formation were altered. Two crystal structures of Dpo4 with a template N{sup 2}-NaphG (in a post-insertion register opposite a 3'-terminal C in the primer) were solved. One showed N{sup 2}-NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 'little finger' domain. The Hoogsteen face was within hydrogen bonding distance of the N4 atoms of the cytosine opposite N{sup 2}-NaphG and the cytosine at the -2 position. The second structure showed N{sup 2}-Naph G in an anti conformation with the primer terminus largely disordered. Collectively these results explain the versatility of Dpo4 in bypassing bulky G lesions.

  8. Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part I: bulky DNA adducts.

    PubMed

    Topinka, J; Milcova, A; Libalova, H; Novakova, Z; Rossner, P; Balascak, I; Sram, R J

    2009-10-02

    (32)P-postlabelling and PAH-ELISA using the antiserum #29 were employed to analyze DNA adducts in venous and umbilical cord blood and the placenta of 79 mothers giving birth to 80 living babies in Prague (Czech Republic). Ambient air exposure was measured by stationary measurements of basic air pollutants (PM2.5, c-PAHs) during the entire pregnancy. Tobacco smoke exposure was assessed by questionnaire data and by plasma cotinine levels. The total DNA adduct levels in the lymphocytes of mothers and newborns were elevated by 30-40% (p<0.001) compared with the placenta. B[a]P-like DNA adduct (adduct with the identical chromatographic mobility on TLC as major BPDE derived DNA adduct) levels were elevated in the blood of mothers compared with the placenta and the blood of newborns (p<0.05 and p<0.01). In tobacco smoke-exposed mothers, higher DNA adduct levels in the blood of mothers and newborns compared with the placenta were found (p<0.001), whereas the total and B[a]P-like adduct levels were comparable in the blood of mothers and newborns. B[a]P-like adducts were elevated in the blood of mothers unexposed to tobacco smoke compared with that of corresponding newborns and the placenta (p<0.01). Total and B[a]P-like DNA adducts were increased in the placenta of tobacco smoke-exposed compared with unexposed mothers (p<0.001 and p<0.01). In lymphocytes of tobacco smoke-exposed mothers, the comparison of total adduct levels (1.18+/-0.67 vs. 0.92+/-0.28) and B[a]P-like DNA adducts (0.22+/-0.12 adducts/10(8) nucleotides vs. 0.15+/-0.06 adducts/10(8) nucleotides) with newborns indicated a 30-40% increase of adducts in mothers. Almost equal PAH-DNA adduct levels were detected by anti-BPDE-DNA ELISA in the placenta of tobacco smoke-exposed and -unexposed mothers. Our results suggest a protective effect of the placental barrier against the genotoxic effect of some tobacco smoke components between the circulation of mother and child. We found a correlation between adduct levels

  9. Bulky DNA adducts in human sperm associated with semen parameters and sperm DNA fragmentation in infertile men: a cross-sectional study

    PubMed Central

    2013-01-01

    Background DNA adducts are widely used marker of DNA damage induced by environmental pollutants. The present study was designed to explore whether sperm polycyclic aromatic hydrocarbon-DNA adducts were associated with sperm DNA integrity and semen quality. Methods A total of 433 Han Chinese men were recruited from an infertility clinic. Immunofluorescence was applied to analyze sperm PAH-DNA adducts. Sperm DNA fragmentation was detected by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labelling (TUNEL) assay. Results After adjustment for potential confounders using linear regression, sperm PAH-DNA adducts were negatively associated with sperm concentration, total sperm count, sperm motility, and curvilinear velocity (VCL). In addition, a positive relationship between sperm PAH-DNA adducts and sperm DNA fragmentation was found. Conclusions Our findings suggested an inverse association between sperm PAH-DNA adducts and semen quality, and provided the first epidemiologic evidence of an adverse effect of PAH-DNA adducts on sperm DNA integrity. PMID:24073787

  10. Insight into mechanisms of 3′-5′ exonuclease activity and removal of bulky 8,5′-cyclopurine adducts by apurinic/apyrimidinic endonucleases

    PubMed Central

    Mazouzi, Abdelghani; Vigouroux, Armelle; Aikeshev, Bulat; Brooks, Philip J.; Saparbaev, Murat K.; Morera, Solange; Ishchenko, Alexander A.

    2013-01-01

    8,5′-cyclo-2'-deoxyadenosine (cdA) and 8,5′-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases—Escherichia coli Xth and human APE1—can remove 5′S cdA (S-cdA) at 3′ termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3′ end. To understand the structural basis of 3′ repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2′-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3′-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3′→5′ exonuclease function and its inhibition by 3′-terminal S-cdA residue. This structure was used as a template to model 3′-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3′ repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3′ DNA termini in E. coli and human cells, respectively. PMID:23898172

  11. The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK.

    PubMed

    Schreck, I; Grico, N; Hansjosten, I; Marquardt, C; Bormann, S; Seidel, A; Kvietkova, D L; Pieniazek, D; Segerbäck, D; Diabaté, S; van der Horst, G T J; Oesch-Bartlomowicz, B; Oesch, F; Weiss, C

    2016-02-18

    Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

  12. [Mass spectrometric analysis of polycyclic aromatic hydrocarbons adducted to DNA]. Final report

    SciTech Connect

    Barofsky, D.F.

    1992-12-31

    Studies described herein sought and to synthesize PAH-adducted residues of DNA to serve as models for carrying out the mass spectrometric studies; to construct and test a high performance, pulsed ion bombardment, time-of-flight (TOF) mass spectrometer; to initiate an investigation of the efficacy of using thin wire sample holders to increase sensitivity and focused ion beam bombardment to increase ion yield and ion transmission; and to initiate an investigation of sensitivity enhancing matrices for PAH-adducted DNA.

  13. sup 14 C-sulfur mustard adducts of calf thymus DNA. Final report, Aug-Sep 90

    SciTech Connect

    Yaverbaum, S.

    1991-02-01

    A grant was awarded to TNO-PML to develop immunochemical monitoring systems for the detection of DNA-HD and Protein-HD adducts in humans following exposure to HD. TNO-PML has been using 35S-HD to prepare adducts for their assays, which have inherent shortcomings that limit detection sensitivity. An experimental batch of 14C-HD-DNA adducts was prepared in an attempt to increase the assay sensitivity. Double - and single-stranded purified calf thymus DNA preparations were reacted with 142, 14.2 and 1.42 uM of 14C-HD under aqueousfree conditions. The 14C-HD-DNA adducts were isolated at -20C in 75% ethanol solution and freed of HD agent and organic solvents (i.e., acetone and alcohol). The 14C-HD-DNA adducts in aqueous buffer were analyzed for specific activity and purity. The ds-DNA-HD adducts were uncontaminated, but the ss-DNA-HD adducts were initially slightly contaminated with alcohol.

  14. Characterization of thioether-linked protein adducts of DNA using a Raney-Ni-mediated desulfurization method and liquid chromatography-electrospray-tandem mass spectrometry.

    PubMed

    Chowdhury, Goutam; Guengerich, F Peter

    2015-03-09

    This unit contains a complete procedure for the detection and structural characterization of DNA protein crosslinks (DPCs). The procedure also describes an approach for the quantitation of the various structurally distinct DPCs. Although various methods have been described in the literature for labile DPCs, characterization of nonlabile adducts remain a challenge. Here we present a novel approach for characterization of both labile and non-labile adducts by the use of a combination of chemical, enzymatic, and mass spectrometric approaches. A Raney Ni-catalyzed reductive desulfurization method was used for removal of the bulky peptide adducts, enzymatic digestion was used to digest the protein to smaller peptides and DNA to nucleosides, and finally LC-ESI-tandem mass spectrometry (MS) was utilized for detection and characterization of nucleoside adducts. Copyright © 2015 John Wiley & Sons, Inc.

  15. Sperm DNA oxidative damage and DNA adducts

    PubMed Central

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  16. The importance of carcinogen dose in chemoprevention studies: quantitative interrelationships between, dibenzo[a,l]pyrene dose, chlorophyllin dose, target organ DNA adduct biomarkers and final tumor outcome.

    PubMed

    Pratt, M Margaret; Reddy, Ashok P; Hendricks, Jerry D; Pereira, Cliff; Kensler, Thomas W; Bailey, George S

    2007-03-01

    Chlorophyllin (CHL) is a potent antimutagen in vitro, an effective anti-carcinogen in several animal models, and significantly reduced urinary biomarkers of aflatoxin B(1) (AFB(1)) exposure in a human population. Here we report an expanded analysis of CHL chemoprevention using the potent environmental hydrocarbon dibenzo[a,l]pyrene (DBP). A dose-dose matrix design employed over 12 000 rainbow trout to evaluate the interrelationships among dietary carcinogen dose, anti-carcinogen dose, carcinogen-DNA adduct levels at exposure and eventual tumor outcome in two target organs. Included was an evaluation of the pharmaceutical CHL preparation (Derifil), used previously in a study of individuals chronically exposed to AFB(1). CHL was pre-, co- and post-fed at doses of 0-6000 p.p.m. and co-fed with DBP at doses of 0-371.5 p.p.m. for 4 weeks. This protocol generated a total of 21 dose-dose treatment groups, each evaluated with three or more replicates of 100 animals. The DBP-only treatment produced dose-responsive increases in liver and stomach DBP-DNA adducts, whereas increasing CHL co-treatment doses produced successive inhibition in liver (49-83%) and stomach (47-75%) adduct levels at each DBP dose examined. The remaining 8711 trout were necropsied, 10 months later. DBP treatment alone produced a logit incidence versus log [DBP] dose-response curve in stomach that was linear; CHL co-treatment provided dose-dependent tumor inhibition which ranged from 30 to 68% and was predictable from the adduct response. The Derifil CHL preparation was also found to effectively reduce DNA adduction and final tumor incidence in stomach (as well as liver), with a potency compatible with its total chlorin content. Liver tumor incidence in the DBP-only groups appeared to plateau near 60%. At DBP doses of adducts as biomarkers. At 225 p.p.m. DBP, however, very high CHL doses were

  17. POOLED ANALYSIS OF STUDIES ON DNA ADDUCTS AND DIETARY VITAMINS

    PubMed Central

    Ragin, Camille; Minor, Aerie; Agudo, Antonio; Farmer, Peter; Garte, Seymour; Gonzales, Carlos; Kalina, Ivan; Matullo, Pino; Popov, Todor; Palli, Domenico; Peluso, Marco; Riccieri, Fulvio; Sram, Radim; Vineis, Paolo; Taioli, Emanuela

    2010-01-01

    Objectives There is some evidence that dietary components that are rich in antioxidant and vitamins are inversely associated with DNA adduct levels induced by environmental carcinogens such as polycyclic aromatic hydrocarbons, although the epidemiologic data are inconsistent. This study addresses the association between vitamins, DNA adducts and smoking. Methods A combined analysis of individual data on the association between bulky DNA adducts and dietary vitamins were conducted. A Medline search was performed to identify studies on healthy subjects in which smoking and vitamins intake information were available, and bulky DNA adducts were measured in peripheral blood with 32P post labeling. Eight published studies met the eligibility criteria, and individual data from 7 data sets including 2,758 subjects were obtained. GSTM1 and GSTT1 were also available on all the subjects. Results Vitamin E was inversely significantly associated with DNA adducts after adjustment for possible confounding factors. Vitamin A and C were not independent predictors of DNA adducts. A stratified analysis showed that vitamin A had a significant inverse association with DNA adducts in ever smokers only. Conclusions This result is relevant to planning any future chemo-preventive interventions directed to high risk subgroups of the population, for cancer prevention. PMID:20399891

  18. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    DOE PAGES

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; ...

    2015-10-28

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. In this paper, we present the first, to ourmore » knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Finally and hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.« less

  19. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    SciTech Connect

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-10-28

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. In this paper, we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Finally and hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  20. Quantitative inter-relationships between aflatoxin B1 carcinogen dose, indole-3-carbinol anti-carcinogen dose, target organ DNA adduction and final tumor response.

    PubMed

    Dashwood, R H; Arbogast, D N; Fong, A T; Pereira, C; Hendricks, J D; Bailey, G S

    1989-01-01

    A number of recent studies have described inhibitor-mediated reductions in the covalent DNA binding and tumorigenicity of various carcinogens, in species such as rats, mice and rainbow trout (Salmo gairdneri). Since inhibitory effects have, in most cases, been reported after testing at one carcinogen and one inhibitor level only, the detailed relationships between carcinogen dose, inhibitor dose, in vivo DNA binding and final tumor response are not well understood in any species. To determine these relationships we have employed the trout model in a combined DNA binding/tumor dose-response protocol using approximately 10,000 animals. Trout were pretreated with one of five different dose-levels of indole-3-carbinol (I3C), a naturally occurring anti-carcinogen found in cruciferous vegetables such as broccoli and cabbage. After 4 weeks, animals received the same dietary level of I3C for a further 2 weeks together with [3H]aflatoxin B1 (AFB1) in the dose-range 10-320 p.p.b. From tanks containing 150 animals (three tanks per I3C-AFB1 dose-point), 15 fish were selected at random in order to assess hepatic AFB1-DNA binding levels. Remaining animals were returned to control diet for determination of tumor response at 12 months. Linear increases in DNA binding occurred with dose of AFB1 at each I3C dose-level. Successive increases in I3C dose gave dose-related decreases in AFB1-DNA binding, resulting in a series of curves of decreasing slope. Shifts in DNA-binding slopes were compared quantitatively with horizontal displacements towards higher carcinogen dose in corresponding tumor dose-response curves. At I3C doses of less than or equal to 2000 p.p.m., the inhibitor-altered tumor response was predicted precisely by changes in dose received (DNA adducts formed) in the target organ. These data constitute the first direct evidence of pure anti-initiating activity by a natural anti-carcinogen found in human diet, where all animals were treated at the same time and under

  1. Induction of Gter dot C to Ater dot T transitions by the acridine half-mustard ICR-191 supports a mispairing mechanism for mutagenesis by some bulky mutagens

    SciTech Connect

    Sahasrabudhe, S.R.; Luo, Xun; Humayun, M.Z. )

    1990-12-01

    As the most nucleophilic atom in DNA, the guanine N7 atom is a major site of attack for a large number of chemical mutagens as well as chemotherapeutic agents. Paradoxically, while methylation of guanine N7 is believed to be largely nonmutagenic, aflatoxin B{sub 1}, among the most potent mutagens, appears to exert its mutagenic activity through adduction at this site. On the basis of an analysis of the specificity of mutations induced by various adduct forms of aflatoxin B{sub 1}, the authors have previously proposed mechanisms that can both resolve the paradox and account for the specificity of mutagenesis by aflatoxin B{sub 1}. The hypothesized mechanisms specify how a bulky guanine N7 lesion can promote G{center dot}C to A{center dot}T transitions as well as frame-shift mutations. Since the proposed mechanisms are in principle lesion-independent, a simple test of the proposed mechanisms would be to examine the specificity of mutations induced by a structurally different bulky guanine N7 adduct. Toward this goal, M13 replicative form DNA was subjected to in vitro adduction with the acridine mutagen ICR-191 and transfected into Escherichia coli. Mutations in the LacZ({alpha}) gene segments were scored and defined at the sequence level. The results show that ICR-191 adduction induces both base substitutions and frame shifts with near-equal efficiency. A clear majority of base substitutions were G{center dot}C to A{center dot}T transitions. The data presented in this communication are consistent with the proposed mechanisms for base substition mutagenesis by bulky guanine N7 lesions and permit a further refinement of the previously proposed mechanisms for frame-shift induction by bulky DNA lesions.

  2. Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA

    SciTech Connect

    Leadon, S.A.; Hanawalt, P.C.

    1984-11-01

    Excision repair of bulky adducts in alpha DNA of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have found that u.v. irradiation of these cells results in the enhanced removal of both aflatoxin B1 (AFB1) and acetylaminofluorene (AAF) adducts from the alpha DNA sequences without affecting repair in the bulk of the DNA. The degree of enhanced removal of AFB1 is dependent upon the u.v. dose and the time interval between irradiation and AFB1 treatment. The u.v. enhancement is not inhibited by cycloheximide. Exposure of the cells to dimethylsulfate or gamma-rays does not affect AFB1 adduct repair. The formation and removal of N-acetoxy-2-acetylaminofluorene (NA-AAF) adducts from alpha and bulk DNA was studied in detail. A higher initial level of the acetylated C8 adduct of guanine was found in alpha DNA than in bulk DNA. Although both the acetylated and deacetylated C8 adducts were removed from the two DNA species, the level of repair was significantly greater in the bulk DNA. Irradiation of cells with u.v. prior to treatment with NA-AAF enhanced the removal of both adducts from alpha DNA with little or no effect on repair in bulk DNA. We conclude that the presence of u.v. photoproducts or some intermediate in their processing alters the chromatin structure of alpha DNA thereby rendering bulky adducts accessible to repair enzymes. In addition, the differential formation and repair of AAF adducts in alpha DNA compared with that in the bulk of the genome supports the hypothesis of an altered chromatin structure for alpha domains.

  3. The analysis of DNA adducts: The transition from 32P-postlabeling to mass spectrometry

    PubMed Central

    Klaene, Joshua J.; Sharma, Vaneet K.; Glick, James; Vouros, Paul

    2012-01-01

    The technique of 32P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10 μg DNA to achieve the detection of 1 adduct in 1010 normal bases. 32P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques 32P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field. Structural methods have since been developed to allow for positive identification of DNA adducts, but to this day, the same level of sensitivity and low sample requirements provided by 32P-postlabeling have not been matched. In this mini review we will discuss the 32P-postlabeling method and chronicle the transition to mass spectrometry via the hyphenation of gas chromatography, capillary electrophoresis, and ultimately liquid chromatography which, some 30 years later, is only just starting to approach the sensitivity and low sample requirements of 32P-postlabeling. This paper focuses on the detection of bulky carcinogen-DNA adducts, with no mention of oxidative damage or small alkylating agents. This is because the 32P-postlabeling assay is most compatible with bulky DNA adducts. This will also allow a more comprehensive focus on a subject that has been our particular interest since 1990. PMID:22960573

  4. Effects of benzo[a]pyrene adduct stereochemistry on downstream DNA replication in vitro: evidence for different adduct conformations within the active site of DNA polymerase I (Klenow fragment).

    PubMed

    Alekseyev, Yuriy O; Romano, Louis J

    2002-04-02

    The presence of bulky adducts in DNA is known to interfere with DNA replication not only at the site of the lesion but also at positions up to 5 nucleotides past the adduct location. Kinetic studies of primer extension by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) (KF) when (+)-trans- or (+)-cis-B[a]P-N(2)-dG adducts were positioned in the double-stranded region of the primer-templates showed that both stereoisomers significantly block downstream replication. However the (+)-cis adduct, which causes a stronger inhibition of the nucleotides insertion across from and immediately past the lesion, affected the downstream replication to a much smaller extent than did the (+)-trans adduct, especially when the B[a]P-modified dG was properly paired with a dC. The effects of mismatches across from the adduct and the sequence context surrounding the adduct were also dependent on the stereochemistry of the B[a]P adduct. Thus, the identity of the nucleotide across from the adduct that provided the best downstream replication was different for the (+)-cis and (+)-trans adducts, a factor that might differentially contribute to the mutagenic bypass of these lesions. These findings provide strong direct evidence that the conformations of the (+)-cis and (+)-trans adducts within the active site of KF are significantly different and probably differentially affect the interactions of the polymerase with the minor groove, thereby leading to different replication trends. The stereochemistry of the adduct was also found to differentially affect the sequence-mediated primer-template misalignments, resulting in different consequences during the bypass of the lesion.

  5. Bulky waste quantities and treatment methods in Denmark.

    PubMed

    Larsen, Anna W; Petersen, Claus; Christensen, Thomas H

    2012-02-01

    Bulky waste is a significant and increasing waste stream in Denmark. However, only little research has been done on its composition and treatment. In the present study, data about collection methods, waste quantities and treatment methods for bulky waste were obtained from two municipalities. In addition a sorting analysis was conducted on combustible waste, which is a major fraction of bulky waste in Denmark. The generation of bulky waste was found to be 150-250 kg capita(-1) year(-1), and 90% of the waste was collected at recycling centres; the rest through kerbside collection. Twelve main fractions were identified of which ten were recyclable and constituted 50-60% of the total quantity. The others were combustible waste for incineration (30-40%) and non-combustible waste for landfilling (10%). The largest fractions by mass were combustible waste, bricks and tile, concrete, non-combustible waste, wood, and metal scrap, which together made up more than 90% of the total waste amounts. The amount of combustible waste could be significantly reduced through better sorting. Many of the waste fractions consisted of composite products that underwent thorough separation before being recycled. The recyclable materials were in many cases exported to other countries which made it difficult to track their destination and further treatment.

  6. Optically pure bulky (hetero)arylalkyl carbinols via kinetic resolution.

    PubMed

    Hu, Bin; Meng, Meng; Fossey, John S; Mo, Weimin; Hu, Xinquan; Deng, Wei-Ping

    2011-10-14

    Planar chiral nucleophilic catalyst Fc-PIP was employed in the kinetic resolution of bulky (hetero)arylalkyl carbinols delivering unreacted alcohols with extremely high enantiomeric excess (>99.0% ees) in ideal conversions ranging from 50.4-56.7%.

  7. Isolevuglandin Adducts in Disease

    PubMed Central

    Bi, Wenzhao

    2015-01-01

    Abstract Significance: A diverse family of lipid-derived levulinaldehydes, isolevuglandins (isoLGs), is produced by rearrangement of endoperoxide intermediates generated through both cyclooxygenase (COX) and free radical-induced cyclooxygenation of polyunsaturated fatty acids and their phospholipid esters. The formation and reactions of isoLGs with other biomolecules has been linked to alcoholic liver disease, Alzheimer's disease, age-related macular degeneration, atherosclerosis, cardiac arythmias, cancer, end-stage renal disease, glaucoma, inflammation of allergies and infection, mitochondrial dysfunction, multiple sclerosis, and thrombosis. This review chronicles progress in understanding the chemistry of isoLGs, detecting their production in vivo and understanding their biological consequences. Critical Issues: IsoLGs have never been isolated from biological sources, because they form adducts with primary amino groups of other biomolecules within seconds. Chemical synthesis enabled investigation of isoLG chemistry and detection of isoLG adducts present in vivo. Recent Advances: The first peptide mapping and sequencing of an isoLG-modified protein present in human retina identified the modification of a specific lysyl residue of the sterol C27-hydroxylase Cyp27A1. This residue is preferentially modified by iso[4]LGE2 in vitro, causing loss of function. Adduction of less than one equivalent of isoLG can induce COX-associated oligomerization of the amyloid peptide Aβ1-42. Adduction of isoLGE2 to phosphatidylethanolamines causes gain of function, converting them into proinflammatory isoLGE2-PE agonists that foster monocyte adhesion to endothelial cells. Future Directions: Among the remaining questions on the biochemistry of isoLGs are the dependence of biological activity on isoLG isomer structure, the structures and mechanism of isoLG-derived protein–protein and DNA–protein cross-link formation, and its biological consequences. Antioxid. Redox Signal. 22

  8. Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair.

    PubMed Central

    Wei, D; Maher, V M; McCormick, J J

    1996-01-01

    Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide

  9. DNA adduct formation among workers in a Thai industrial estate and nearby residents.

    PubMed

    Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Meunier, Aurelie; Sangrajrang, Suleeporn; Piro, Sara; Ceppi, Marcello; Boffetta, Paolo

    2008-01-25

    The genotoxic effects of air pollutant exposures have been studied in people living and working in Map Ta Phut, Rayong province, Thailand, a site where is located the Map Ta Phut Industrial Estate (MIE) one of the largest steel, refinery and petrochemical complex in the South-Eastern Asia. This was done by the conduction of a transversal study aimed to compare the prevalence of bulky DNA adducts in groups of subjects experiencing various degree of air pollution. DNA adduct analysis was performed in the leukocytes of 201 volunteers by the (32)P-postlabelling assay: 79 were workers in the MIE complex, including 24 refinery workers, 40 steel workers and 15 tinplate workers, 72 were people residing downwind in the MIE area and 50 were residents in a control district of the same Rayong province but without industrial exposures. The groups of workers were analyzed separately to evaluate if DNA adduct formation differs by the type of industry. The levels of bulky DNA adducts were 1.17+/-0.17 (SE) adducts/10(8) nucleotides in refinery workers, 1.19+/-0.19 (SE) in steel workers, 0.87+/-0.17 (SE) in tinplate workers, 0.85+/-0.07 (SE) in MIE residents and 0.53+/-0.05 (SE) in district controls. No effects of smoking habits on DNA adducts was found. The multivariate regression analysis shows that the levels of DNA adducts were significantly increased among the individuals living near the MIE industrial complex in respect to those resident in a control district (p<0.05). In the groups of occupationally exposed workers, the highest levels of DNA adducts were found among the workers experiencing an occupational exposure to polycyclic aromatic hydrocarbons, e.g. the steel factory and refinery workers. When we have evaluated if the levels of DNA adducts of the PAH exposed workers were different from those of the MIE residents, a statistical significantly difference was found (p<0.05). Our present study indicates that people living near point sources of industrial air pollution can

  10. Alcohol, Aldehydes, Adducts and Airways

    PubMed Central

    Sapkota, Muna; Wyatt, Todd A.

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  11. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-11-05

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease.

  12. Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance.

    PubMed

    Weiß, Martin S; Pavlidis, Ioannis V; Spurr, Paul; Hanlon, Steven P; Wirz, Beat; Iding, Hans; Bornscheuer, Uwe T

    2017-06-01

    Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild-type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence-based motif that supports the identification of novel enzymes. However, these variants were not active against 2,2-dimethyl-1-phenyl-propan-1-one, which carries a bulky tert-butyl substituent adjacent to the carbonyl function. Here, we report a solution for this type of substrate. The evolved ATAs perform asymmetric synthesis of the respective R amine with high conversions by using either alanine or isopropylamine as amine donor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Dynamic heat and moisture transfer in bulky PAN nanofiber mats

    NASA Astrophysics Data System (ADS)

    Borhani, Sedigheh; Etemad, Seyed Gholamreza; Ravandi, Seyed Abdolkarim Hosseini

    2011-07-01

    In this study a non-conventional electrospinning technique was designed for the production of high bulky polyacrylonitrile (PAN) nanofiber mats. Optimum nanofiber mats are achieved with 15 wt.% solution of PAN in dimethylformamide. Such mats result in a bulk porosity which is as high as 99.9 and a density as low as 0.84 × 10-3 g/cm3. The effect of the porosity of nanofiber mats on the air permeability and coupled heat and moisture transfer of fibers was investigated. Based on the results, high bulky nanofiber mats possess high heat and moisture transfer. Experimental data reveal that upon a slight decrease in the bulk porosity, air permeability and heat transfer decrease noticeably, while moisture transfer variation is low.

  14. Achievement of Bulky Homochirality in Zeolitic Imidazolate-Related Frameworks.

    PubMed

    Wang, Fei; Tang, Yu-Huan; Zhang, Jian

    2015-12-07

    Before this work, adding chiral C centers into zeolitic imidazolate frameworks (ZIFs) has never been realized. Presented here are the first examples on achieving bulky homochirality in ZIF systems, and three homochiral zeolitic imidazolate-related frameworks with sodalite and dia topologies are successfully synthesized by employing enantiopure imidazolate derivatives. The results open a new blueprint on the synthetic design of homochiral ZIFs for future applications.

  15. Translesion Synthesis Past Acrolein-derived DNA Adducts by Human Mitochondrial DNA Polymerase γ*

    PubMed Central

    Kasiviswanathan, Rajesh; Minko, Irina G.; Lloyd, R. Stephen; Copeland, William C.

    2013-01-01

    Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N2-propano-2′-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N6-propano-2′-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates. PMID:23543747

  16. Should simple hysterectomy be added after chemo-radiation for stage IB2 and bulky IIA cervical carcinoma?

    PubMed

    Eitan, Ram; Levavi, Hanoch; Peled, Yoav; Brenner, Ronen; Sabah, Gad; Ben-Arie, Alon; Dgani, Ram; Fishman, Ami; Sulkes, Aaron; Fenig, Eyal; Koren, Claude

    2010-06-01

    Management of bulky cervical tumours is controversial. We describe the addition of high dose rate brachytherapy with concomitant chemotherapy to an attenuated protocol of radiation followed by simple hysterectomy in the management of bulky cervical tumours. Between January, 2003 and December, 2006, 23 patients diagnosed with bulky cervical tumours underwent a fixed chemo-radiation protocol followed by simple hysterectomy. Fractionated external beam pelvic radiation (4500 cGy) followed by two high-dose rate applications of brachytherapy (700 cGy - prescription dose to point A) was given with weekly concomitant cisplatin (35 mg/m(2)). Patients then underwent simple hysterectomy. Clinical information was prospectively collected and patient charts were then further reviewed. Twenty patients had stage IB2 and three bulky IIA. Median tumour size was 5 cm. Sixteen patients (70%) achieved a clinical complete and seven (30%) a clinical partial response. All patients had a total abdominal hysterectomy and bilateral salpingo-oophorectomy (TAH BSO). On final pathology, 12 patients (52%) had a pathological complete response, whereas 11 patients (48%) had residual carcinoma in the cervix. Surgical margins were not involved. With a median follow-up time of 20 months (range 10-50 months), four patients (17.4%), all from the pathological partial response group, have suffered a pelvic recurrence, within 6 months from therapy; nineteen patients (82.6%) remain free of disease. This attenuated protocol of chemo-radiation using HDR brachytherapy followed by simple hysterectomy is a viable option in the treatment of bulky cervical carcinomas. The rate of residual cervical disease after chemo-radiation is substantial, but simple hysterectomy achieved negative surgical margins in all cases.

  17. A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

    PubMed

    Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

    2012-01-01

    Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein.

  18. Aromatic adducts and lung cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) Spanish cohort.

    PubMed

    Gilberson, Tamra; Peluso, Marco E M; Munia, Armelle; Luján-Barroso, Leila; Sánchez, María-José; Navarro, Carmen; Amiano, Pilar; Barricarte, Aurelio; Quirós, J Ramón; Molina-Montes, Esther; Sánchez-Cantalejo, Emilio; Tormo, María-José; Chirlaque, María-Dolores; Ardanaz, Eva; Dorronsoro, Miren; Confortini, Massimo; Bonet, Catalina; Sala, Núria; González, Carlos A; Agudo, Antonio

    2014-09-01

    In this case-cohort study, we examined the association between bulky DNA adducts and the risk of lung cancer within the European Prospective Investigation into Cancer and Nutrition (EPIC) Spanish cohort with an average 7-year follow-up, including 98 cases of primary lung cancer and 296 subjects randomly selected from the cohort. Aromatic adducts were measured using (32)P-postlabeling in leukocyte DNA from blood samples collected at enrollment. The association between DNA adducts and the risk of lung cancer was estimated using a Cox proportional hazards model with a modified partial likelihood. There was an overall significant increased risk for developing lung cancer when DNA adduct concentrations were doubled, with relative risk (RR) adjusting for all relevant confounders of 1.36 with 95% confidence interval (CI) 1.18-157. There was a significant increased risk for developing lung cancer when DNA adduct concentrations were doubled for current smokers and among subjects exposed to PAH at work; there was also a slightly higher increase among males than females. However, no statistically significant differences were observed for the effect of adduct levels across smoking status, sex or occupational exposure to PAH. A meta-analysis combined four prospective studies, including this study, resulting in a significant association among current smokers, with an overall estimate of 34% increase in the risk of lung cancer when doubling the level of aromatic DNA adducts in leukocytes.

  19. Real-time surface plasmon resonance study of biomolecular interactions between polymerase and bulky mutagenic DNA lesions.

    PubMed

    Xu, Lifang; Vaidyanathan, V G; Cho, Bongsup P

    2014-10-20

    Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl (FABP) or N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5'-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo(-)) or polymerase β (pol β). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo(-) binds strongly to a double strand/single strand template/primer junction, whereas pol β binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol β, Kf-exo(-) binds tightly to lesion-modified templates; however, both polymerases exhibited minimal nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and (19)F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared to that in CG*A. Although Kf-exo(-) was not sensitive to the presence of a DNA lesion, FAAF-induced conformational heterogeneity perturbed the active site of pol β, weakening the enzyme's ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.

  20. Efficiency of bulky protic solvent for SN2 reaction.

    PubMed

    Lee, Sung-Sik; Kim, Ho-Sung; Hwang, Tae-Kyu; Oh, Young-Ho; Park, Sung-Woo; Lee, Sungyul; Lee, Byoung Se; Chi, Dae Yoon

    2008-01-03

    We calculate and compare the effects of aprotic vs protic solvent on the rate of SN2 reaction [F- + C3H7OMs--> C3H7F + OMs-]. We find that aprotic solvent acetonitrile is more efficient than a small protic solvent such as methanol. Bulky protic solvent (tert-butyl alcohol) is predicted to be quite efficient, giving the rate constant that is similar to that in CH3CN. Our calculated relative activation barriers of the SN2 reaction in methanol, tert-butyl alcohol, and CH3CN are in good agreement with experimental observations.

  1. Preparation of scaffolds based on bulky sutures for cell therapy

    NASA Astrophysics Data System (ADS)

    Park, Young Hwan; Chun, Heung Jae; Kim, Sung Jin

    2012-04-01

    Scaffold for cell therapy was prepared with poly (lactide-co-glicolide, PLA/PGA (10:90). By using melt-spinning and draw texturing process, we could prepare microfibrous bulky suture which had heterogeneous macropore. Microfibrous structure has great potentiality as biomimicking architecture for cell growth and maintaining cell functions. The result of cell seeding showed that pore size, pore distribution, and fiber fineness of sutures were suitable as a biocompatible scaffold in vitro for NIH 3T3 Fibroblast cell. Also, we expect that prepared scaffold for cell-therapy will provide numerous benefits as a noninvasive alternative for tissue engineering applications.

  2. Preparation of scaffolds based on bulky sutures for cell therapy

    NASA Astrophysics Data System (ADS)

    Park, Young Hwan; Chun, Heung Jae; Kim, Sung Jin

    2011-11-01

    Scaffold for cell therapy was prepared with poly (lactide-co-glicolide, PLA/PGA (10:90). By using melt-spinning and draw texturing process, we could prepare microfibrous bulky suture which had heterogeneous macropore. Microfibrous structure has great potentiality as biomimicking architecture for cell growth and maintaining cell functions. The result of cell seeding showed that pore size, pore distribution, and fiber fineness of sutures were suitable as a biocompatible scaffold in vitro for NIH 3T3 Fibroblast cell. Also, we expect that prepared scaffold for cell-therapy will provide numerous benefits as a noninvasive alternative for tissue engineering applications.

  3. Regiochemically controlled synthesis of a β-4-β' [70]fullerene bis-adduct

    DOE PAGES

    Cerón, Maira R.; Castro, Edison; Neti, Venkata S. Pavan K.; ...

    2016-12-22

    A β-4-β' C70 bis-adduct regioisomer and an uncommon mono-adduct β-malonate C70 derivative were synthesized by using a Diels–Alder cycloaddition followed by an addition–elimination of bromo-ethylmalonate and a retro-Diels–Alder cycloaddition reaction. Here, we also report the regioselective synthesis and spectroscopic characterization of Cs-symmetric tris- and C2v-symmetric tetra-adducts of C70, which are the precursors of the mono- and bis-adduct final products.

  4. Regiochemically controlled synthesis of a β-4-β' [70]fullerene bis-adduct

    SciTech Connect

    Cerón, Maira R.; Castro, Edison; Neti, Venkata S. Pavan K.; Dunk, Paul W.; Echegoyen, Luis A.

    2016-12-22

    A β-4-β' C70 bis-adduct regioisomer and an uncommon mono-adduct β-malonate C70 derivative were synthesized by using a Diels–Alder cycloaddition followed by an addition–elimination of bromo-ethylmalonate and a retro-Diels–Alder cycloaddition reaction. Here, we also report the regioselective synthesis and spectroscopic characterization of Cs-symmetric tris- and C2v-symmetric tetra-adducts of C70, which are the precursors of the mono- and bis-adduct final products.

  5. Effect of Watson-Crick and Hoogsteen base pairing on the conformational stability of C8-phenoxyl-2'-deoxyguanosine adducts.

    PubMed

    Millen, Andrea L; Churchill, Cassandra D M; Manderville, Richard A; Wetmore, Stacey D

    2010-10-14

    Bulky DNA addition products (adducts) formed through attack at the C8 site of guanine can adopt the syn orientation about the glycosidic bond due to changes in conformational stability or hydrogen-bonding preferences directly arising from the bulky group. Indeed, the bulky substituent may improve the stability of (non-native) Hoogsteen pairs. Therefore, such adducts often result in mutations upon DNA replication. This work examines the hydrogen-bonded pairs between the Watson-Crick and Hoogsteen faces of the ortho or para C8-phenoxyl-2'-deoxyguanosine adduct and each natural (undamaged) nucleobase with the goal to clarify the conformational preference of this type of damage, as well as provide insight into the likelihood of subsequent mutation events. B3LYP/6-311+G(2df,p)//B3LYP/6-31G(d) hydrogen-bond strengths were determined using both nucleobase and nucleoside models for adduct pairs, as well as the corresponding complexes involving natural 2'-deoxyguanosine. In addition to the magnitude of the binding strengths, the R(C1'···C1') distances and ∠(N9C1'C1') angles, as well as the degree of propeller-twist and buckle distortions, were carefully compared to the values observed in natural DNA strands. Due to structural changes in the adduct monomer upon inclusion of the sugar moiety, the monomer deformation energy significantly affects the relative hydrogen-bond strengths calculated with the nucleobase and nucleoside models. Therefore, we recommend the use of at least a nucleoside model to accurately evaluate hydrogen-bond strengths of base pairs involving flexible, bulky nucleobase adducts. Our results also emphasize the importance of considering both the magnitude of the hydrogen-bond strength and the structure of the base pair when predicting the preferential binding patterns of nucleobases. Using our best models, we conclude that the Watson-Crick face of the ortho phenoxyl adduct forms significantly more stable complexes than the Hoogsteen face, which

  6. Catalytic behaviour in the ring-opening polymerisation of organoaluminiums supported by bulky heteroscorpionate ligands.

    PubMed

    Castro-Osma, Jose A; Alonso-Moreno, Carlos; Lara-Sánchez, Agustín; Otero, Antonio; Fernández-Baeza, Juan; Sánchez-Barba, Luis F; Rodríguez, Ana M

    2015-07-21

    A series of alkyl organoaluminium complexes based on bulky heteroscorpionate ligands were designed as catalysts for the ring-opening polymerisation of cyclic esters. Thus, the treatment of AlX3 (X = Me, Et) with bulky acetamide or thioacetamide heteroscorpionate ligands nbptamH (1) [nbptamH = N-naphthyl-2,2-bis(3,5-dimethylpyrazol-1-yl)thioacetamide], fbpamH (2) [fbpamH = N-fluorenyl-2,2-bis(3,5-dimethylpyrazol-1-yl)acetamide], ptbptamH (3) [ptbptamH = N-phenyl-2,2-bis(3,5-di-tert-butylpyrazol-1-yl)thioacetamide], ntbptamH (4) [ntbptamH = N-naphthyl-2,2-bis(3,5-di-tert-butylpyrazol-1-yl)thioacetamide], ptbpamH (5) [ptbpamH = N-phenyl-2,2-bis(3,5-di-tert-butylpyrazol-1-yl)acetamide] and (S)-mtbpamH (6) [(S)-mtbpamH = (S)-(−)-N-α-methylbenzyl-2,2-bis(3,5-di-tert-butylpyrazol-1-yl)acetamide] for 1 hour at 0 °C afforded the dialkyl aluminium complexes [AlX2{κ(2)-nbptam}] (X = Me 7, Et 8), [AlX2{κ(2)-fbpam}] (X = Me 9, Et 10), [AlX2{κ(2)-ptbptam}] (X = Me 11, Et 12), [AlX2{κ(2)-ntbptam}] (X = Me 13, Et 14), [AlX2{κ2(-)ptbpam}] (X = Me 15, Et 16) and [AlX2{κ(2)-(S)-mtbpam}] (X = Me 17, Et 18). The structures of the complexes were determined by spectroscopic methods and the X-ray crystal structure of 14 was also established. The alkyl-containing aluminium complexes 7–18 can act as efficient single-component initiators for the ring-opening polymerisation of ε-caprolactone and rac-lactide. The polymerisations are living, as evidenced by the narrow polydispersities of the isolated polymers and the linear nature of the number average molecular weight versus conversion plot. Finally, a comparative study of ring-opening polymerisation for new bulky heteroscorpionate aluminium initiators and the less congested aluminium analogues is reported.

  7. Biomarkers for exposure to ambient air pollution--comparison of carcinogen-DNA adduct levels with other exposure markers and markers for oxidative stress.

    PubMed

    Autrup, H; Daneshvar, B; Dragsted, L O; Gamborg, M; Hansen, M; Loft, S; Okkels, H; Nielsen, F; Nielsen, P S; Raffn, E; Wallin, H; Knudsen, L E

    1999-03-01

    Human exposure to genotoxic compounds present in ambient air has been studied using selected biomarkers in nonsmoking Danish bus drivers and postal workers. A large interindividual variation in biomarker levels was observed. Significantly higher levels of bulky carcinogen-DNA adducts (75.42 adducts/10(8) nucleotides) and of 2-amino-apidic semialdehyde (AAS) in plasma proteins (56.7 pmol/mg protein) were observed in bus drivers working in the central part of Copenhagen, Denmark. In contrast, significantly higher levels of AAS in hemoglobin (55.8 pmol/mg protein), malondialdehyde in plasma (0. 96 nmol/ml plasma), and polycyclic aromatic hydrocarbon (PAH)-albumin adduct (3.38 fmol/ microg albumin) were observed in the suburban group. The biomarker levels in postal workers were similar to the levels in suburban bus drivers. In the combined group of bus drivers and postal workers, negative correlations were observed between bulky carcinogen-DNA adduct and PAH-albumin levels (p = 0.005), and between DNA adduct and [gamma]-glutamyl semialdehyde (GGS) in hemoglobin (p = 0.11). Highly significant correlations were found between PAH-albumin adducts and AAS in plasma (p = 0.001) and GGS in hemoglobin (p = 0.001). Significant correlations were also observed between urinary 8-oxo-7, 8-dihydro-2'-deoxyguanosine and AAS in plasma (p = 0.001) and PAH-albumin adducts (p = 0.002). The influence of the glutatione S-transferase (GST) M1 deletion on the correlation between the biomarkers was studied in the combined group. A significant negative correlation was only observed between bulky carcinogen-DNA adducts and PAH-albumin adducts (p = 0.02) and between DNA adduct and urinary mutagenic activity (p = 0.02) in the GSTM1 null group, but not in the workers who were homozygotes or heterozygotes for GSTM1. Our results indicate that some of the selected biomarkers can be used to distinguish between high and low exposure to environmental genotoxins.

  8. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    NASA Astrophysics Data System (ADS)

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-11-01

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  9. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Parsons, Zachary D; Yuen, Philip K; David, Sheila S; Igarashi, Yasuhiro; Eichman, Brandt F

    2015-11-12

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  10. The N(2)-Furfuryl-deoxyguanosine Adduct Does Not Alter the Structure of B-DNA.

    PubMed

    Ghodke, Pratibha P; Gore, Kiran R; Harikrishna, S; Samanta, Biswajit; Kottur, Jithesh; Nair, Deepak T; Pradeepkumar, P I

    2016-01-15

    N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by ∼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct.

  11. Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I.

    PubMed

    Zhuang, P; Kolbanovskiy, A; Amin, S; Geacintov, N E

    2001-06-05

    The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the

  12. Molecular evidence of the involvement of the nucleotide excision repair (NER) system in the repair of the mono(ADP-ribosyl)ated DNA adduct produced by pierisin-1, an apoptosis-inducing protein from the cabbage butterfly.

    PubMed

    Kawanishi, Masanobu; Matsukawa, Kazuki; Kuraoka, Isao; Takamura-Enya, Takeji; Totsuka, Yukari; Matsumoto, Yasuko; Watanabe, Masahiko; Zou, Yue; Tanaka, Kiyoji; Sugimura, Takashi; Wakabayashi, Keiji; Yagi, Takashi

    2007-04-01

    Pierisin-1 is a potent apoptosis-inducing protein found in the pupal extract of the cabbage white butterfly. Pierisin-1 catalyzes the mono(ADP-ribosyl)ation of the 2'-deoxyguanosine residue and produces a bulky adduct, N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG) in DNA. Here, we examined the involvement of the nucleotide excision repair (NER) system in the removal of N2-ADPR-dG in Escherichia coli (E. coli) and human cells. The results of mobility shift gel electrophoresis assays using a 50-mer oligodeoxynucleotide containing a single N2-ADPR-dG showed that E. coli UvrAB proteins bound to the N2-ADPR-dG in vitro. Incubation of the adducted oligodeoxynucleotides with UvrABC resulted in the incision of the oligonucleotides in vitro. The results of filter binding and gel mobility shift assays using human XPA protein showed that XPA bound to DNA containing N2-ADPR-dGs in vitro. Finally, we introduced plasmids containing N2-ADPR-dGs into E. coli and human cells. N2-ADPR-adducted plasmids replicated l0 times and 20 times less efficiently in NER-deficient E. coli and human cells than in their wild-type counterparts, respectively. More mutations were induced in the plasmid propagated in NER-deficient cells than that in wild-type human cells. These results indicate the involvement of the NER system in the repair of N2-ADPR-dG in both E. coli and human cells.

  13. Mass Spectrometric Detection of Sulfur Mustard Adducts to Proteins and DNA: Dosimetry of Exposure to Sulfur Mustard

    DTIC Science & Technology

    1993-05-13

    agents. In this context, we developed immunochemical analysis procedures for sulfur mustard adducts to DNA which allow to detect exposure of human...We did not succeed so far in developing a suitable immunochemical method for detection of sulfur mustard-protein adducts . Presently, we are extending...Verification, dosimetry and biomonitoring of mustard gas exposure via immunochemical detection of mustard gas adducts to DNA and proteins. Final

  14. A bulky dermoid cyst of the floor of the mouth.

    PubMed

    Boko, E; Amaglo, K; Kpemissi, E

    2014-04-01

    Bulky dermoid cysts of the floor of the mouth are very rare and may pose a problem of diagnosis. They also raise problems for the anesthesiologist and surgeon. We report the first case to be described in Togo. A 23-year-old man was admitted for a submental submandibular sublingual mass. It was soft, depressable, painless, without adenopathies, raising the tongue against the palate and creating a "second tongue-like" aspect. Resection on intra-oral route removed an intact cyst of 13cm long axis. Histology diagnosed dermoid cyst. Dermoid cysts of the floor of the mouth present as a submental sublingual mass, which may cause dyspnea and disorders of swallowing, chewing and/or vocal function. Differential diagnosis concerns sublingual, submental and cervical masses. Definitive diagnosis is founded on the histology specimen. Imaging may assist diagnosis. Intubation may be problematic. The resection approach may be intra-oral or cervical. Dermoid cysts of the floor of the mouth are rare. They may induce functional disorder. An intra-oral approach is preferable when possible. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  15. Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase iota: Hoogsteen or Watson-Crick base pairing?

    PubMed

    Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

    2009-01-13

    Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase iota (poliota) is a bypass polymerase of the Y family. Crystal structures of poliota suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that poliota is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in poliota for bypass of dG-AAF. In poliota with Hoogsteen-paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick-paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that poliota would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for poliota in lesion bypass.

  16. Aromatic DNA adducts and polymorphisms in metabolic genes in healthy adults: findings from the EPIC-Spain cohort.

    PubMed

    Agudo, Antonio; Peluso, Marco; Sala, Núria; Capellá, Gabriel; Munnia, Armelle; Piro, Sara; Marín, Fátima; Ibáñez, Raquel; Amiano, Pilar; Tormo, M José; Ardanaz, Eva; Barricarte, Aurelio; Chirlaque, M Dolores; Dorronsoro, Miren; Larrañaga, Nerea; Martínez, Carmen; Navarro, Carmen; Quirós, J Ramón; Sánchez, M José; González, Carlos A

    2009-06-01

    Aromatic compounds such as polycyclic aromatic hydrocarbons, arylamines and heterocyclic amines require metabolic activation to form metabolites able to bind to DNA, a process mediated by polymorphic enzymes. We measured aromatic DNA adducts in white blood cells by the (32)P-post-labelling assay in a sample of 296 healthy adults (147 men and 149 women) from five regions of Spain. We also analyzed functional polymorphisms in the metabolic genes CYP1A1, CYP1A2, EPHX1, GSTM1, GSTT1, NAT2 and SULT1A1. A significant increased level of DNA aromatic adducts was found related to the fast oxidation-hydrolysis phenotype defined by the polymorphism I462V in CYP1A1, the allele A in IVS1-154C>A of CYP1A2 and the combination Tyrosine-Arginine for Y113H and H139R of EPHX1. Geometric means (adducts per 10(-9) normal nucleotides) were 2.17, 4.04 and 6.30 for slow, normal and fast phenotypes, respectively (P-trend = 0.01). Slow acetylation by NAT2 was associated with a significant decrease in adduct level; subjects with slow alleles *5A and *7A/B had in average 1.56 x 10(-9)adducts, as compared with 5.60 for those with normal NAT2 activity (P-value = 0.01). No association was seen with polymorphisms of other metabolic genes such as GSTM1, GSTT1 or SULT1A1. We concluded that the metabolic pathways of oxidation, hydrolysis and acetylation are relevant to the formation of bulky DNA adducts. This could suggest a potential involvement of aromatic compounds in the formation of such adducts; however, given lack of specificity of the post-labeling assay, a firm conclusion cannot be drawn.

  17. Aromaticity/Bulkiness of Surface Ligands to Promote the Interaction of Anionic Amphiphilic Gold Nanoparticles with Lipid Bilayers.

    PubMed

    Gao, Jinhong; Zhang, Ouyang; Ren, Jing; Wu, Chuanliu; Zhao, Yibing

    2016-02-16

    The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity.

  18. Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons

    PubMed Central

    Georgiadis, Panagiotis; Kovács, Katalin; Kaila, Stella; Makedonopoulou, Paraskevi; Anna, Livia; Poirier, Miriam C.; Knudsen, Lisbeth E.; Schoket, Bernadette; Kyrtopoulos, Soterios A.

    2012-01-01

    We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)–DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE–DNA SCIA has a limit of detection of 3 adducts per 109 nucleotides with 5 μg DNA per well. We have validated the BPDE–DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30–60%) than levels of bulky DNA adducts measured in the same samples by 32P-postlabelling. The BPDE–DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE–DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between 32P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies. PMID:22610669

  19. Late-onset bulky naevocytoma of the perineum masquerading as a malignant melanoma.

    PubMed

    Huh, J W; Yoo, J; Kim, M S; Choi, K H; Jue, M S; Park, H J

    2017-03-01

    Bulky naevocytoma of the perineum is a very rare variant of giant congenital melanocytic naevus (GCMN). It presents as a bulky naevocytic tumour in the perineal region with characteristic histological findings, such as extensive areas with a neural appearance called 'lames foliacees', formation of a pseudofollicular structure and extension of naevus cells between collagen bundles in a row called 'Indian-file' pattern. We report a case of late-onset bulky naevocytoma of the perineum in a 13-year-old girl. The patient presented with two bulky, pedunculated, heavily pigmented masses in the vulvar area that developed in a pre-existing GCMN lesion, which began around puberty and caused severe gait disturbance. Given the possibility of malignant transformation, we conducted staged reduction surgery of the tumour masses, which were found to be intradermal naevi without evidence of malignancy. The patient's gait disturbance improved markedly after surgery.

  20. Protein adduct biomarkers: State of the Art

    SciTech Connect

    Meyer, M.J.; Bechtold, W.E.

    1996-10-01

    Covalent protein adducts formed after exposure to xenobiotics may provide readily measurable indicators of these exposures, After adequate characterization of the dose-dependent formation of a specific adduct, the adduct can often be used as a quantitative marker for exposure, DNA adduct formation, or, possibly, risk of disease. By elucidating the structure of an adduct and studying the conditions under which it forms, information about the reactions that lead to its formation can be obtained. Continuing work in this area includes methods to expand the number, types, and levels of chemical exposures that can be studied by covalent adduct formation. In addition to the use of this technology in the field of occupational health, basic research in this area provides insights into metabolic pathways and biochemistry, as well. 26 refs., 1 fig.

  1. Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

    SciTech Connect

    Shi, Yun-bo

    1988-03-01

    This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs.

  2. An integrated QSAR-PBK/D modelling approach for predicting detoxification and DNA adduct formation of 18 acyclic food-borne α,β-unsaturated aldehydes.

    PubMed

    Kiwamoto, R; Spenkelink, A; Rietjens, I M C M; Punt, A

    2015-01-01

    Acyclic α,β-unsaturated aldehydes present in food raise a concern because the α,β-unsaturated aldehyde moiety is considered a structural alert for genotoxicity. However, controversy remains on whether in vivo at realistic dietary exposure DNA adduct formation is significant. The aim of the present study was to develop physiologically based kinetic/dynamic (PBK/D) models to examine dose-dependent detoxification and DNA adduct formation of a group of 18 food-borne acyclic α,β-unsaturated aldehydes without 2- or 3-alkylation, and with no more than one conjugated double bond. Parameters for the PBK/D models were obtained using quantitative structure-activity relationships (QSARs) defined with a training set of six selected aldehydes. Using the QSARs, PBK/D models for the other 12 aldehydes were defined. Results revealed that DNA adduct formation in the liver increases with decreasing bulkiness of the molecule especially due to less efficient detoxification. 2-Propenal (acrolein) was identified to induce the highest DNA adduct levels. At realistic dietary intake, the predicted DNA adduct levels for all aldehydes were two orders of magnitude lower than endogenous background levels observed in disease free human liver, suggesting that for all 18 aldehydes DNA adduct formation is negligible at the relevant levels of dietary intake. The present study provides a proof of principle for the use of QSAR-based PBK/D modelling to facilitate group evaluations and read-across in risk assessment. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. 32P-postlabeling assay for the quantification of the major platinum-DNA adducts.

    PubMed

    Pluim, D; Maliepaard, M; van Waardenburg, R C; Beijnen, J H; Schellens, J H

    1999-11-01

    To allow more sensitive, selective, and routine analyses of platinum(Pt)-GG and -AG intrastrand cross-links we have significantly improved our quantitative (32)P-postlabeling assay (M. J. P. Welters et al. Carcinogenesis 18, 1767-1774, 1997). Instead of off-line scintillation counting we introduced an on-line flow radioisotope detector into the HPLC system. Furthermore, the isolation protocol for the adducts was significantly modified and optimized to reduce interfering background peaks that prevented quantification of low levels of the cisplatin-DNA adducts in white blood cells obtained from patients. Reduction of background signals was obtained by boiling the samples, followed by phenol/chloroform/isoamylethanol extraction after the DNA digestion step. The labeling efficiency for the adducts was increased by 40% by using Na-formate instead of NH(4)-formate for elution of the adducts from the strong cation-exchange columns. Finally, a calibration curve and quality controls were implemented. The labeling efficiencies were not different between the dinucleotides. The between- and within-run precision for the Pt-GG and Pt-AG adducts measured at the lower limit of quantification of 87 and 53 amol/microg DNA, respectively, was less than 20% CV. The adducts were stable in DNA stored for a 2-month time period at -80 degrees C. The assay is now routinely used for high-precision analyses of patient and cell line samples containing very low adduct levels. Copyright 1999 Academic Press.

  4. Synthesis of polycyclic aromatic hydrocarbon adducts of adenine in nucleosides and oligonucleotides

    SciTech Connect

    Kim, S.J.

    1992-01-01

    PAHs are activated metabolically to electrophilic intermediates such as diol epoxides, which bind to cellular macromolecules, particularly DNA. The dominant paradigm for the etiology of chemically induced cancer involves alterations in the structure of DNA which can cause replication errors. Synthesis of oligonucleotides bearing regiochemically and stereochemically defined PAH adducts is a prerequisite to any study of the relationships of adduct structure to mutagenicity. The first chapter introduces the history of PAH carcinogenesis studies. The second chapter contains the synthetic methodology for preparation of deoxyadenosine adducts of PAHs: benzo[a]pyrene, benz[a]anthracene, and benzo[c]phenanthrene. The nucleoside adducts were synthesized efficiently via the reaction of racemic aminotriol derivatives of PAHs with 6-fluoropurine deoxyriboside. The enantiopure PAH adducts of deoxyadenosine were obtained by HPLC separation of the diastereomers which were characterized by [sup 1]H and 2D NMR, CD, UV, and Mass spectroscopy. The third chapter describes syntheses of oligonucleotides bearing regiochemically and stereochemically defined PAH adducts of benzo[a]pyrene and benz[a]anthracene. A post-oligomerization strategy was used in which the condensation reaction between the aminotriol and a fluoronucleoside was carried out after assembling of the oligomer but before the final deprotection step. The fourth chapter describes an efficient synthesis of (+) and ([minus]) benzo[a]pyrene adducts of deoxyadenosine 3[prime]-phosphate which will be valuable as authentic standards in the Randerath [sup 32]P postlabeling procedure for identification of DNA adducts. In chapter five, a synthetic approach to PAH adducts of deoxyguanosine is discussed. A selective deprotection method is explored in which the oligonucleotide is assembled in the normal fashion except for the exocyclic amino group of the target nucleotide being protected with a labile protecting group.

  5. Bilateral failure of adduction following orbital decompression.

    PubMed Central

    Kinsella, F; Kyle, P; Stansfield, A

    1990-01-01

    We report a case of bilateral complete failure of adduction following bilateral translid antralethmoidal orbital decompression. We believe the probable mechanism is neuropraxia (temporary dysfunction) of the third cranial nerves' supply to the medial recti, owing to these nerves' occupying an anatomically abnormal position. Partial recovery of adduction occurred over the ensuing six months. Images PMID:2337551

  6. Site-specific aflatoxin B sub 1 adduction of sequence-positioned nucleosome core particles

    SciTech Connect

    Moyer, R.A.

    1988-01-01

    The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. This problem was circumvented by constructing nucleosomes that are homogenous in DNA-histone contact points. A cloned DNA fragment, containing a sea urchin 5S gene which precisely positions a histone octamer was employed. By using {sup 32}P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate (DMS) and the bulky alkylating agent afatoxin B{sub 1}-dichloride (AFB{sub 1}-Cl{sub 2}) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in sequence-positioned core particles.

  7. DNA adducts induced by the potential human carcinogen cyclopenta[cd]pyrene using [sup 32]P-postlabeling

    SciTech Connect

    Beach, A.C.

    1993-01-01

    Cyclopenta[cd]pyrene (CPP) is a widespread polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. CPP is of interest for its prevalence in such notable sources of human exposure to environmental pollution as automotive/diesel engine exhaust emissions. As DNA adduct formation represents an integral phase in the chemical carcinogenesis process, the following studies were undertaken to determine: (1) the utility [sup 32]P-postlabeling for detecting CPP-DNA adducts; (2) the potency and efficacy of CPP to induce DNA adducts in vivo; and (3) the identity of the ultimate mutagenic intermediate(s) of CPP in vivo. CPP-3,4-epoxide has been suggested as the ultimate mutagenic form of CPP. This epoxide was reacted with DNA and deoxynucleotide induced with those formed by CPP and the base-specificity of those adducts. CPP-3,4-epoxide induced at least four major and three minor DNA adducts at the level of deoxyguanosine. All adducts were similar to those formed by CPP in pilot in vivo studies; therefore the ability of CPP to induce adducts was further evaluated. CPP produced up to four major and seven minor adducts in the rat. CPP-DNA adducts were highest in the lung followed by the heart, WBCs, and liver. CPP was also found to induce adducts in vivo across a five orders of magnitude dosing range i.e. 1 [mu]g/kg-50 mg/kg. CPP adducts induced in vitro were found to increase in the presence of epoxide hydrolase inhibitors suggesting their epoxide origin. This prompted evaluation of the similarity of adducts induced by CPP in vivo with those produced by CPP-3,4-epoxide in vitro. Cochromatographic studies revealed that all four major and at least five of the minor adducts induced by CPP were formed by reaction of CPP-3,4-epoxide with deoxyguanosine; one adduct was clearly oxyadenosine derived. Finally, [sup 32]P-postlabeling was used to detect CPP-adducts in assays with potential for human application.

  8. Bulky Dehydroamino Acids Enhance Proteolytic Stability and Folding in β-Hairpin Peptides.

    PubMed

    Jalan, Ankur; Kastner, David W; Webber, Kei G I; Smith, Mason S; Price, Joshua L; Castle, Steven L

    2017-10-06

    The bulky dehydroamino acids dehydrovaline (ΔVal) and dehydroethylnorvaline (ΔEnv) can be inserted into the turn regions of β-hairpin peptides without altering their secondary structures. These residues increase proteolytic stability, with ΔVal at the (i + 1) position having the most substantial impact. Additionally, a bulky dehydroamino acid can be paired with a d-amino acid (i.e., d-Pro) to synergistically enhance resistance to proteolysis. A link between proteolytic stability and peptide structure is established by the finding that a stabilized ΔVal-containing β-hairpin is more highly folded than its Asn-containing congener.

  9. Relaxation spectra and dipolar correlations for flexible polymers with bulky side groups

    SciTech Connect

    Diaz-Calleja, R.; Riande, E.; Roman, J.S.

    1992-08-06

    This paper discusses how relaxation spectra and dipolar correlations for flexible polymers with bulky side groups (PBPA chains) suggest that intermolecular correlations are not very important in this polymer and that {alpha}, {beta}, and {gamma} absorptions exist. TSDC techniques reveal that the {gamma} peak has a smaller activation energy than the {beta}, and the coupling scheme is used to interpret the complex dielectric and mechanical {alpha} relaxations. The anomalous temperature dependence of the glass-rubber relaxation is discussed in terms of the bulkiness of the side group. 23 refs., 8 figs., 3 tabs.

  10. Mechanism of Error-Free Bypass of the Environmental Carcinogen N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase η.

    PubMed

    Patra, Amritraj; Politica, Dustin A; Chatterjee, Arindom; Tokarsky, E John; Suo, Zucai; Basu, Ashis K; Stone, Michael P; Egli, Martin

    2016-11-03

    The environmental pollutant 3-nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8-dG-ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error-free manner by the human Y-family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol⋅DNA⋅dCTP complex between a C8-dG-ABA-containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error-free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra-nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson-Crick pair with the incoming dCTP.

  11. Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase ι: Hoogsteen or Watson-Crick base pairing?†

    PubMed Central

    Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

    2009-01-01

    Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase ι (polι) is a bypass polymerase of the Y family. Crystal structures of polι suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that polι is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in polι for bypass of dG-AAF. In polι with Hoogsteen paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that polι would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for polι in lesion bypass. PMID:19072536

  12. Implications of acetaldehyde-derived DNA adducts for understanding alcohol-related carcinogenesis.

    PubMed

    Balbo, Silvia; Brooks, Philip J

    2015-01-01

    Among various potential mechanisms that could explain alcohol carcinogenicity, the metabolism of ethanol to acetaldehyde represents an obvious possible mechanism, at least in some tissues. The fundamental principle of genotoxic carcinogenesis is the formation of mutagenic DNA adducts in proliferating cells. If not repaired, these adducts can result in mutations during DNA replication, which are passed on to cells during mitosis. Consistent with a genotoxic mechanism, acetaldehyde does react with DNA to form a variety of different types of DNA adducts. In this chapter we will focus more specifically on N2-ethylidene-deoxyguanosine (N2-ethylidene-dG), the major DNA adduct formed from the reaction of acetaldehyde with DNA and specifically highlight recent data on the measurement of this DNA adduct in the human body after alcohol exposure. Because results are of particular biological relevance for alcohol-related cancer of the upper aerodigestive tract (UADT), we will also discuss the histology and cytology of the UADT, with the goal of placing the adduct data in the relevant cellular context for mechanistic interpretation. Furthermore, we will discuss the sources and concentrations of acetaldehyde and ethanol in different cell types during alcohol consumption in humans. Finally, in the last part of the chapter, we will critically evaluate the concept of carcinogenic levels of acetaldehyde, which has been raised in the literature, and discuss how data from acetaldehyde genotoxicity are and can be utilized in physiologically based models to evaluate exposure risk.

  13. Detection and characterization of DNA adducts formed from metabolites of the fungicide ortho-phenylphenol.

    PubMed

    Zhao, Shouxun; Narang, Amarjit; Gierthy, John; Eadon, George

    2002-05-22

    The significance of DNA adduction in ortho-phenylphenol-induced carcinogenesis remains unclear. Establishing adduct structures may contribute to resolving this issue. The chemical structures of the DNA adduction products resulting from the in vitro reaction of phenylbenzoquinone, the putative ultimate carcinogenic metabolite of the fungicide/disinfectant ortho-phenylphenol, are reported here. Three isomeric adducts that resulted from reaction of deoxyguanosine were characterized by UV, LC-ESI-MS, and MS/MS, and 1D and 2D COSY-NMR spectroscopy. The proposed mechanism of product formation is nucleophilic attack by the deoxyguanosine exocyclic amine nitrogen on an electrophilic quinone carbon, followed by stabilization through enolization. Another nucleophilic attack forms a five-membered ring, which aromatizes by dehydration to form the final product. Adducts were also characterized from deoxyadenosine and deoxycytidine, although conversions were at least 10 times lower. Structures are also proposed for these products. Cell culture studies confirmed that HepG2 cells incubated with phenylbenzoquinone at concentrations associated with cytotoxicity form the same DNA adducts.

  14. Acetaldehyde and the genome: beyond nuclear DNA adducts and carcinogenesis.

    PubMed

    Brooks, Philip J; Zakhari, Samir

    2014-03-01

    The designation of acetaldehyde associated with the consumption of alcoholic beverages as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer (IARC) has brought renewed attention to the biological effects of acetaldehyde, as the primary oxidative metabolite of alcohol. Therefore, the overall focus of this review is on acetaldehyde and its direct and indirect effects on the nuclear and mitochondrial genome. We first consider different acetaldehyde-DNA adducts, including a critical assessment of the evidence supporting a role for acetaldehyde-DNA adducts in alcohol related carcinogenesis, and consideration of additional data needed to make a conclusion. We also review recent data on the role of the Fanconi anemia DNA repair pathway in protecting against acetaldehyde genotoxicity and carcinogenicity, as well as teratogenicity. We also review evidence from the older literature that acetaldehyde may impact the genome indirectly, via the formation of adducts with proteins that are themselves critically involved in the maintenance of genetic and epigenetic stability. Finally, we note the lack of information regarding acetaldehyde effects on the mitochondrial genome, which is notable since aldehyde dehydrogenase 2 (ALDH2), the primary acetaldehyde metabolic enzyme, is located in the mitochondrion, and roughly 30% of East Asian individuals are deficient in ALDH2 activity due to a genetic variant in the ALDH2 gene. In summary, a comprehensive understanding of all of the mechanisms by which acetaldehyde impacts the function of the genome has implications not only for alcohol and cancer, but types of alcohol related pathologies as well.

  15. Smoking-related DNA adducts as potential diagnostic markers of lung cancer: new perspectives.

    PubMed

    Grigoryeva, E S; Kokova, D A; Gratchev, A N; Cherdyntsev, E S; Buldakov, M A; Kzhyshkowska, J G; Cherdyntseva, N V

    2015-03-01

    In recent years, the new direction such as identification of informative circulating markers reflecting molecular genetic changes in the DNA of tumor cells was actively developed. Smoking-related DNA adducts are very promising research area, since they indicate high pathogenetic importance in the lung carcinogenesis and can be identified in biological samples with high accuracy and reliability using highly sensitive mass spectrometry methods (TOF/TOF, TOF/MS, MS/MS). The appearance of DNA adducts in blood or tissues is the result of the interaction of carcinogenic factors, such as tobacco constituents, and the body reaction which is determined by individual characteristics of metabolic and repair systems. So, DNA adducts may be considered as a cumulative mirror of heterogeneous response of different individuals to smoking carcinogens, which finally could determine the risk for lung cancer. This review is devoted to analysis of the role of DNA adducts in lung carcinogenesis in order to demonstrate their usefulness as cancer associated markers. Currently, there are some serious limitations impeding the widespread use of DNA adducts as cancer biomarkers, due to failure of standardization of mass spectrometry analysis in order to correctly measure the adduct level in each individual. However, it is known that all DNA adducts are immunogenic, their accumulation over some threshold concentration leads to the appearance of long-living autoantibodies. Thus, detection of an informative pattern of autoantibodies against DNA adducts using innovative multiplex ELISA immunoassay may be a promising approach to find lung cancer at an early stage in high-risk groups (smokers, manufacturing workers, urban dwellers).

  16. Turning the Page: Forget about Those Bulky Backbreakers, Digital Textbooks Are the Future

    ERIC Educational Resources Information Center

    Hill, Rebecca

    2010-01-01

    Remember when computers were mostly used in offices? They were big and bulky with about as much mobility as a beached whale. Forget about using them in the classroom. Forget about reading a book on them. Forget about an app, well, for anything. Today, computers are the number-one educational tool. In fact, no one can imagine a school without one.…

  17. Turning the Page: Forget about Those Bulky Backbreakers, Digital Textbooks Are the Future

    ERIC Educational Resources Information Center

    Hill, Rebecca

    2010-01-01

    Remember when computers were mostly used in offices? They were big and bulky with about as much mobility as a beached whale. Forget about using them in the classroom. Forget about reading a book on them. Forget about an app, well, for anything. Today, computers are the number-one educational tool. In fact, no one can imagine a school without one.…

  18. An integrated QSAR-PBK/D modelling approach for predicting detoxification and DNA adduct formation of 18 acyclic food-borne α,β-unsaturated aldehydes

    SciTech Connect

    Kiwamoto, R. Spenkelink, A.; Rietjens, I.M.C.M.; Punt, A.

    2015-01-01

    Acyclic α,β-unsaturated aldehydes present in food raise a concern because the α,β-unsaturated aldehyde moiety is considered a structural alert for genotoxicity. However, controversy remains on whether in vivo at realistic dietary exposure DNA adduct formation is significant. The aim of the present study was to develop physiologically based kinetic/dynamic (PBK/D) models to examine dose-dependent detoxification and DNA adduct formation of a group of 18 food-borne acyclic α,β-unsaturated aldehydes without 2- or 3-alkylation, and with no more than one conjugated double bond. Parameters for the PBK/D models were obtained using quantitative structure–activity relationships (QSARs) defined with a training set of six selected aldehydes. Using the QSARs, PBK/D models for the other 12 aldehydes were defined. Results revealed that DNA adduct formation in the liver increases with decreasing bulkiness of the molecule especially due to less efficient detoxification. 2-Propenal (acrolein) was identified to induce the highest DNA adduct levels. At realistic dietary intake, the predicted DNA adduct levels for all aldehydes were two orders of magnitude lower than endogenous background levels observed in disease free human liver, suggesting that for all 18 aldehydes DNA adduct formation is negligible at the relevant levels of dietary intake. The present study provides a proof of principle for the use of QSAR-based PBK/D modelling to facilitate group evaluations and read-across in risk assessment. - Highlights: • Physiologically based in silico models were made for 18 α,β-unsaturated aldehydes. • Kinetic parameters were determined by in vitro incubations and a QSAR approach. • DNA adduct formation was negligible at levels relevant for dietary intake. • The use of QSAR-based PBK/D modelling facilitates group evaluations and read-across.

  19. Outcomes After Chemotherapy Followed by Radiation for Stage IIB Hodgkin Lymphoma With Bulky Disease.

    PubMed

    Reddy, Jay P; Akhtari, Mani; Smith, Grace L; Pinnix, Chelsea; Osborne, Eleanor M; Gunther, Jillian R; Allen, Pamela K; Yehia, Zeinab Abou; Fanale, Michelle; Rodriguez, M Alma; Fowler, Nathan; Milgrom, Sarah A; Wogan, Christine F; Dabaja, Bouthaina S

    2015-11-01

    Treatment of stage IIB bulky Hodgkin lymphoma (HL) is controversial, with treatment varying by institution. We evaluated patients with IIB bulky disease treated with combined-modality therapy at our institution by describing their long-term outcomes. We identified 149 consecutive patients with stage IIB bulky HL treated between 1971 and 2012. Clinical, pathologic, and treatment characteristics were extracted from medical records. Actuarial overall survival (OS) and relapse-free survival (RFS) were calculated by the Kaplan-Meier method. Independent factors associated with these outcomes were identified by a multivariate Cox regression model. Outcomes were further compared against comparison groups of both advanced-stage and stage IIB patients treated between 1971 and 2009. The 8-year OS rate for patients with stage IIB bulky disease who received combined-modality ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) and radiation was 88.8%; the 8-year RFS rate was 76.8%. On multivariate analysis, age < 40 years (hazard ratio [HR], 0.28; 95% confidence interval [CI], 0.14-0.57; P = .001), receipt of ABVD (vs. MOPP [mechlorethamine, vincristine, procarbazine, prednisone]; HR, 0.32; 95% CI, 0.10-0.88; P = .028), and radiation dose ≥ 30.1 Gy (HR, 0.25; 95% CI, 0.11-0.65; P = .006) were associated with improved OS. Cardiac events (n = 11) and secondary malignancies (n = 11) only occurred in patients treated before 1995. A subgroup analysis demonstrated significantly improved survival in IIB bulky versus advanced-stage patients (8-year OS, 73.4% vs. 57.4%; P = .008). Improved outcomes in patients with in IIB bulky disease were especially evident in the modern era (> 1995; P = .004). Patients with stage IIB bulky HL had excellent outcomes after combined-modality therapy. Treatment strategies have changed substantially over time, with concomitant improvements in disease outcomes and long-term toxicities. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Detection of DNA adducts by bioluminescence

    NASA Astrophysics Data System (ADS)

    Xu, Shunqing; Tan, Xianglin; Yao, Qunfeng; He, Min; Zhou, Yikai; Chen, Jian

    2001-09-01

    Luminescent assay for detection ATP is very sensitive with limitation of 10-17 moles. ATP using styrene oxide as a model carcinogen we currently apply a luminescence technique to detect the very low levels of carcinogen-DNA adducts in vitro and in vivo. The bioluminescent assay of DNA adducts entails three consecutive steps: digestion of modified DNA to adducted dinucleoside monophosphate and normal nucleotide are hydrolyzed to nucleosides (N) by nuclease P1 and prostatic acid phosphomonesterase (PAP); incorporation of (gamma) -P of ATP into normal nucleoside(N); detection of consumption of ATP by luminescence. This assay does not require separate manipulation because of the selective property of nuclease P1. One fmol of carcinogen- DNA adducts was detected by luminescent assay. A good correlation between results of luminescent assay and 32P-postlabeling procedures has been observed. We detect 1 adduct in 108 nucleotides for 10(mu) g DNA sample. The procedures of luminescent method is very simple and low- cost. IT appears applicable to the ultra sensitive detection of low levels of DNA adducts without radioactive isotope.

  1. Modelling informally collected quantities of bulky waste and reusable items in Austria

    SciTech Connect

    Ramusch, R. Pertl, A.; Scherhaufer, S.; Schmied, E.; Obersteiner, G.

    2015-10-15

    Highlights: • Informal collectors from Hungary collect bulky waste and reusable items in Austria. • Two methodologies were applied to estimate the informally collected quantities. • Both approaches lead to an estimation of roughly 100,000 t p.a. informally collected. • The formal Austrian system collects 72 kg/cap/yr of bulky waste, WEE & scrap metal. • Informal collection amounts to approx. 12 kg/cap/yr. - Abstract: Disparities in earnings between Western and Eastern European countries are the reason for a well-established informal sector actively involved in collection and transboundary shipment activities from Austria to Hungary. The preferred objects are reusable items and wastes within the categories bulky waste, WEEE and metals, intended to be sold on flea markets. Despite leading to a loss of recyclable resources for Austrian waste management, these informal activities may contribute to the extension of the lifetime of certain goods when they are reused in Hungary; nevertheless they are discussed rather controversially. The aim of this paper is to provide objective data on the quantities informally collected and transhipped. The unique activities of informal collectors required the development and implementation of a new set of methodologies. The concept of triangulation was used to verify results obtained by field visits, interviews and a traffic counting campaign. Both approaches lead to an estimation of approx. 100,000 t per year of reusable items informally collected in Austria. This means that in addition to the approx. 72 kg/cap/yr formally collected bulky waste, bulky waste wood, household scrap (excluding packaging) and WEEE, up to a further 12 kg/cap/yr might, in the case that informal collection is abandoned, end up as waste or in the second-hand sector.

  2. Antiproliferative activity of the Michael adducts of aroylacrylic acids and cyclic amines.

    PubMed

    Juranić, Ivan O; Tošić, Ana V; Kolundžija, Branka; Drakulić, Branko J

    2014-08-01

    Antiproliferative activity of twenty one Michael adducts of aroylacrylic acids and cyclic amines (N-Me-piperazine, imidazole, 2-Me-imidazole, and indole) was tested toward five human tumor cell lines (HeLa, LS174, K562, FemX, MDA-MB-361) in vitro. Compounds exerted antiproliferative activity in the high to the single-digit micromolar concentrations, causing increase of the cell population fraction in S phase and apoptosis. N-Me-piperazine and imidazole derivatives of aroylacrylic acids substituted with bulky alkyl substituents (2,4-di-i-Pr-Ph-, 2,4,6-tri-Et-Ph-, or β-tetrahydronaphthyl-) showed the best potency, while indole adducts were proved as the inferior antiproliferative agents. Few compounds showed significant selectivity, tumor versus healthy cells, with selectivity index ~60 for the most selective congener. An unbiased in silico distinction between more and less potent compounds was obtained from 3D QSAR models derived by alignment-independent GRIND-2 descriptors.

  3. γ-Selective Cross-Coupling of Allylic Silanolate Salts with Aromatic Bromides Using Trialkylphosphonium Tetrafluoroborate Salts Prepared Directly from Phosphine•Borane Adducts

    PubMed Central

    Denmark, Scott E.; Werner, Nathan S.

    2011-01-01

    The γ-selective, palladium-catalyzed cross-coupling of sodium (Z)-2-butenyldiethylsilanolate with a variety of aromatic bromides is reported. The protocol provides high yields (73–94%) and site-selectivity (γ/α, 25:1–>99:1) in the coupling of electron-rich, electron-poor, sterically hindered, and heteroaromatic bromides. The use of a configurationally homogeneous (Z)-silanolate, nontransferable ethyl groups, and a sterically bulky trialkylphosphonium tetrafluoroborate salt (t-BuCy2PH+BF4−) prepared directly from the corresponding air-stable phosphine•borane adduct are critical to the success of the method. PMID:21830766

  4. Stereotactic Radiosurgery as Part of Multimodal Treatment in a Bulky Leptomeningeal Recurrence of Breast Cancer

    PubMed Central

    Burton, Eric C; Shaughnessy, Joseph N

    2016-01-01

    Breast cancer metastatic to the brain and/or leptomeningeal spread of disease is a frequently encountered clinical situation, especially given the extended course of disease in these patients. Systemic therapies can often effectively prolong extracranial disease control, making effective strategies to control central nervous system-based disease even more critical. We present a case of bulky leptomeningeal relapse of breast cancer in the setting of prior whole brain radiation therapy. In order to treat the patient’s bulky disease and leptomeningeal spread while avoiding the potential toxicities of repeat whole brain radiation, the patient was treated with frameless stereotactic radiosurgery and intrathecal chemotherapy. This is the first report of this treatment approach for leptomeningeal relapse of breast cancer. The patient had an excellent response to treatment and durable intracranial control. PMID:27081584

  5. Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

    PubMed Central

    Liu, Yang; Reeves, Dara; Kropachev, Konstantin; Cai, Yuqin; Ding, Shuang; Kolbanovskiy, Marina; Kolbanovskiy, Alexander; Bolton, Judith L.; Broyde, Suse; Van Houten, Bennett; Geacintov, Nicholas E.

    2011-01-01

    Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend towards similar relative NER incision efficiencies for ~ 65% of these substrates; the other cases deviate mostly by ~ 30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER. PMID:21741328

  6. Endogenous versus Exogenous DNA Adducts: Their Role in Carcinogenesis, Epidemiology, and Risk Assessment

    PubMed Central

    Swenberg, James A.; Moeller, Benjamin C.; Gao, Lina; Upton, Patricia B.; Nakamura, Jun; Starr, Thomas B.

    2011-01-01

    There is a strong need for science-based risk assessment that utilizes known data from diverse sources to arrive at accurate assessments of human health risk. Such assessments will protect the public health without mandating unreasonable regulation. This paper utilizes 30 years of research on three “known human carcinogens”: formaldehyde, vinyl chloride (VC), and ethylene oxide (EO), each of which forms DNA adducts identical to endogenous DNA adducts in all individuals. It outlines quantitative data on endogenous adducts, mutagenicity, and relationships between endogenous and exogenous adducts. Formaldehyde has the richest data set, with quantitative data on endogenous and exogenous DNA adducts from the same samples. The review elaborates on how such data can be used to inform the current risk assessment on formaldehyde, including both the biological plausibility and accuracy of projected risks. Finally, it extends the thought process to VC, EO, and additional areas of potential research, pointing out needs, nuances, and potential paths forward to improved understanding that will lead to strong science-based risk assessment. PMID:21163908

  7. Shape matters: lifecycle of cooperative patches promotes cooperation in bulky populations.

    PubMed

    Misevic, Dusan; Frénoy, Antoine; Lindner, Ariel B; Taddei, François

    2015-03-01

    Natural cooperative systems take many forms, ranging from one-dimensional cyanobacteria arrays to fractal-like biofilms. We use in silico experimental systems to study a previously overlooked factor in the evolution of cooperation, physical shape of the population. We compare the emergence and maintenance of cooperation in populations of digital organisms that inhabit bulky (100 × 100 cells) or slender (4 × 2500) toroidal grids. Although more isolated subpopulations of secretors in a slender population could be expected to favor cooperation, we find the opposite: secretion evolves to higher levels in bulky populations. We identify the mechanistic explanation for the shape effect by analyzing the lifecycle and dynamics of cooperator patches, from their emergence and growth, to invasion by noncooperators and extinction. Because they are constrained by the population shape, the cooperator patches expand less in slender than in bulky populations, leading to fewer cooperators, less public good secretion, and generally lower cooperation. The patch dynamics and mechanisms of shape effect are robust across several digital cooperation systems and independent of the underlying basis for cooperation (public good secretion or a cooperation game). Our results urge for a greater consideration of population shape in the study of the evolution of cooperation across experimental and modeling systems.

  8. Shape matters: Lifecycle of cooperative patches promotes cooperation in bulky populations

    PubMed Central

    Misevic, Dusan; Frénoy, Antoine; Lindner, Ariel B; Taddei, François

    2015-01-01

    Natural cooperative systems take many forms, ranging from one-dimensional cyanobacteria arrays to fractal-like biofilms. We use in silico experimental systems to study a previously overlooked factor in the evolution of cooperation, physical shape of the population. We compare the emergence and maintenance of cooperation in populations of digital organisms that inhabit bulky (100 × 100 cells) or slender (4 × 2500) toroidal grids. Although more isolated subpopulations of secretors in a slender population could be expected to favor cooperation, we find the opposite: secretion evolves to higher levels in bulky populations. We identify the mechanistic explanation for the shape effect by analyzing the lifecycle and dynamics of cooperator patches, from their emergence and growth, to invasion by noncooperators and extinction. Because they are constrained by the population shape, the cooperator patches expand less in slender than in bulky populations, leading to fewer cooperators, less public good secretion, and generally lower cooperation. The patch dynamics and mechanisms of shape effect are robust across several digital cooperation systems and independent of the underlying basis for cooperation (public good secretion or a cooperation game). Our results urge for a greater consideration of population shape in the study of the evolution of cooperation across experimental and modeling systems. PMID:25639379

  9. Modelling informally collected quantities of bulky waste and reusable items in Austria.

    PubMed

    Ramusch, R; Pertl, A; Scherhaufer, S; Schmied, E; Obersteiner, G

    2015-10-01

    Disparities in earnings between Western and Eastern European countries are the reason for a well-established informal sector actively involved in collection and transboundary shipment activities from Austria to Hungary. The preferred objects are reusable items and wastes within the categories bulky waste, WEEE and metals, intended to be sold on flea markets. Despite leading to a loss of recyclable resources for Austrian waste management, these informal activities may contribute to the extension of the lifetime of certain goods when they are reused in Hungary; nevertheless they are discussed rather controversially. The aim of this paper is to provide objective data on the quantities informally collected and transhipped. The unique activities of informal collectors required the development and implementation of a new set of methodologies. The concept of triangulation was used to verify results obtained by field visits, interviews and a traffic counting campaign. Both approaches lead to an estimation of approx. 100,000 t per year of reusable items informally collected in Austria. This means that in addition to the approx. 72 kg/cap/yr formally collected bulky waste, bulky waste wood, household scrap (excluding packaging) and WEEE, up to a further 12 kg/cap/yr might, in the case that informal collection is abandoned, end up as waste or in the second-hand sector.

  10. Repair of furocoumarin adducts in mammalian cells

    SciTech Connect

    Zolan, M.E.; Smith, C.A.; Hanawalt, P.C.

    1984-12-01

    DNA repair was studied in cultured mammalian cells treated with the furocoumarins 8-methoxypsoralen (8-MOP), aminomethyl trioxsalen, or angelicin and irradiated with near UV light. The amount of DNA cross-linked by 8-MOP in normal human cells decreased by about one-half in 24 hours after treatment; no decrease was observed in xeroderma pigmentosum cells, group A. At present, it is not known to what extent this decrease represents complete repair events at the sites of cross-links. Furocoumarin adducts elicited excision repair in normal human and monkey cells but not in xeroderma pigmentosum group A cells. This excision repair resembled in several aspects that elicited by pyrimidine dimers, formed in DNA by irradiation with 254-nm UV light; however, it appeared that for at least 8-MOP and aminomethyl trioxsalen, removal of adducts was not as efficient as was the removal of pyrimidine dimers. A comparison was also made of repair in the 172-base-pair repetitive alpha-DNA component of monkey cells to repair in the bulk of the genome. Although repair elicited by pyrimidine dimers in alpha-DNA was the same as in the bulk DNA, that following treatment of cells with either aminomethyl trioxsalen or angelicin and near UV was markedly deficient in alpha-DNA. This deficiency reflected the removal of fewer adducts from alpha-DNA after the same initial adduct frequencies. These results could mean that each furocoumarin may produce several structurally distinct adducts to DNA in cells and that the capacity of cellular repair systems to remove these various adducts may vary greatly.

  11. Synthesis of Oligodeoxynucleotides Containing a C8-2'-Deoxyguanosine Adduct Formed by the Carcinogen 3-Nitrobenzanthrone.

    PubMed

    Chatterjee, Arindom; Malik, Chanchal K; Basu, Ashis K

    2017-06-19

    This unit describes the detailed procedure in five parts for the synthesis of the C8-2'-deoxyguanosine-3-aminobenzanthrone adduct located in a desired site in an oligonucleotide. The synthesis of the protected 2'-deoxyguanosine, O(6) -benzyl-N(2) -DMTr-3'-5'-bisTBDMS-C8-Br-2'-deoxyguanosine, is described in the first part. The synthesis of the reduced carcinogen 3-aminobenzanthrone is detailed in part two. The third part outlines the key step of the adduct formation between the reduced carcinogen and the protected nucleoside by a palladium-catalyzed cross coupling reaction. The final two parts describe phosphoramidite synthesis from the nucleoside-carcinogen adduct followed by its site-specific incorporation into DNA by solid-phase oligonucleotide synthesis. The adducted oligonucleotides are purified by reversed-phase HPLC and characterized by mass spectrometry. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  12. Relationships between DNA adduct formation and carcinogenesis

    SciTech Connect

    Swenberg, J.A.; Richardson, F.C.; Boucheron, J.A.; Dyroff, M.C.

    1985-10-01

    An impressive array of evidence has been obtained during the past decade establishing correlations between specific DNA adducts and carcinogenesis. Many of the studies utilized organ specific differences in carcinogenesis to establish the correlations. More recently, we have investigated similar relationships between target and nontarget cell populations within the liver. Chronic exposure to methylating hepatocarcinogens predominantly induces hemangiosarcomas, whereas exposure to ethylating agents causes hepatocellular carcinomas. This cell specificity in carcinogenesis correlates well with the presence of promutagenic DNA adducts. In the case of methylating agents, the nonparenchymal cells accumulate O6-methylguanine whereas the hepatocytes do not. Exposure to ethylating agents leads to accumulation of O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine in hepatocytes. These differences reflect the ability of the two cell populations to repair O6-alkylguanine and the extent of purine and pyrimidine alkylation with methylating and ethylating agents. Hepatocytes of rats exposed to diethylnitrosamine for 28 days have four to five times more promutagenic DNA adducts (O6-alkyldeoxyguanosine and O4-alkyldeoxythymidine) than hepatocytes of rats exposed to nearly equimolar doses of dimethylhydrazine. Both O6-methylguanine and O4-methyldeoxythymidine are rapidly repaired by rat hepatocytes, while only O6-ethyldeoxyguanosine is rapidly repaired. Studies comparing the relationship between the induction of gamma-glutamyl transpeptidase-positive foci, hepatocellular carcinoma and promutagenic lesions such as O4-ethyldeoxythymidine will be useful in understanding associations between the molecular dosimetry of DNA adducts, initiation, and progression of hepatocarcinogenesis.

  13. NITRO MUSK ADDUCTS OF RAINBOW TROUT ...

    EPA Pesticide Factsheets

    Rainbow trout and other fish species can serve as 'sentinel' species for the assessment of ecological status and the presence of certain environmental contaminants. As such they act as bioindicators of exposure. Here we present seminal data regarding dose-response and toxicokinetics of trout hemoglobin adduct formation from exposure to nitro musks that are frequently used as fragrance ingredients in formulations of personal care products. Hemoglobin adducts serve as biomarkers of exposure of the sentinel species as we have shown in previous studies of hemoglobin adducts formed in trout and environmental carp exposed to musk xylene (MX) and musk ketone (MK). Gas chromatography-electron capture negative ion chemical ionization-mass spectrometry (GC-NICI-MS) employing selected ion monitoring is used to measure 4-amino-MX (4-AMX), 2-amino-MX (2-AMX), and 2-amino-MK (2-AMK) released by alkaline hydrolysis from the sulfinamide adducts of hemoglobin. Dose-response and toxicokinetics were investigated using this sensitive method for analysis of these metabolites. In the dose-response investigation, the concentrations of 4-AMX and 2-2AMX are observed to pass through a maximum at 0.10 mg/g. In the case of 2-AMK, the adduct concentration is almost the same at dosages in the range of 0.030 to 0.10 mg/g. For toxicokinetics, the concentration of the metabolites in the Hb reaches a maximum in the 3-day sample after administration of MX or MK. Further elimination of the metabo

  14. The rate of homolysis of adducts of peroxynitrite to the C=O double bond.

    PubMed

    Merényi, Gábor; Lind, Johan; Goldstein, Sara

    2002-01-09

    Nucleophilic addition of the peroxynitrite anion, ONOO(-), to the two prototypical carbonyl compounds, acetaldehyde and acetone, was investigated in the pH interval 7.4-14. The process is initiated by fast equilibration between the reactants and the corresponding tetrahedral adduct anion, the equilibrium being strongly shifted to the reactant side. The adduct anion also undergoes fast protonation by water and added buffers. Consequently, the rate of the bimolecular reaction between ONOO(-) and the carbonyl is strongly dependent on the pH and on the concentration of the buffer. The pK(a) of the carbonyl-ONOO adduct was estimated to be approximately 11.8 and approximately 12.3 for acetone and acetaldehyde, respectively. It is shown that both the anionic and the neutral adducts suffer fast homolysis along the weak O-O bond to yield free alkoxyl and nitrogen dioxide radicals. The yield of free radicals was determined to be about 15% with both carbonyl compounds at low and high pH, while the remainder collapses to molecular products in the solvent cage. The rate constants for the homolysis of the adducts vary from ca. 3 x 10(5) to ca. 5 x 10(6) s(-1), suggesting that they cannot act as oxidants in biological systems. This small variation around a mean value of about 10(6) s(-1) suggests that the O-O bond in the adduct is rather insensitive to its protonation state and to the nature of its carbonyl precursor. An overall reaction scheme was proposed, and all the corresponding rate constants were evaluated. Finally, thermokinetic considerations were employed to argue that the formation of dioxirane as an intermediate in the reaction of ONOO(-) with acetone is an unlikely process.

  15. Spectroscopic characterization and modeling of quadrupolar charge-transfer dyes with bulky substituents.

    PubMed

    Sissa, Cristina; Terenziani, Francesca; Painelli, Anna; Siram, Raja Bhaskar Kanth; Patil, Satish

    2012-04-26

    Joint experimental and theoretical work is presented on two quadrupolar D-π-A-π-D chromophores characterized by the same bulky donor (D) group and two different central cores. The first chromophore, a newly synthesized species with a malononitrile-based acceptor (A) group, has a V-shaped structure that makes its absorption spectrum very broad, covering most of the visible region. The second chromophore has a squaraine-based core and therefore a linear structure, as also evinced from its absorption spectra. Both chromophores show an anomalous red shift of the absorption band upon increasing solvent polarity, a feature that is ascribed to the large, bulky structure of the molecules. For these molecules, the basic description of polar solvation in terms of a uniform reaction field fails. Indeed, a simple extension of the model to account for two independent reaction fields associated with the two molecular arms quantitatively reproduces the observed linear absorption and fluorescence as well as fluorescence anisotropy spectra, fully rationalizing their nontrivial dependence on solvent polarity. The model derived from the analysis of linear spectra is adopted to predict nonlinear spectra and specifically hyper-Rayleigh scattering and two-photon absorption spectra. In polar solvents, the V-shaped chromophore is predicted to have a large HRS response in a wide spectral region (approximately 600-1300 nm). Anomalously large and largely solvent-dependent HRS responses for the linear chromophores are ascribed to symmetry lowering induced by polar solvation and amplified in this bulky system by the presence of two reaction fields.

  16. Efficient uptake of dimethyl sulfoxide by the desoxomolybdenum(IV) dithiolate complex containing bulky hydrophobic groups.

    PubMed

    Hasenaka, Yuki; Okamura, Taka-aki; Onitsuka, Kiyotaka

    2015-04-07

    A desoxomolybdenum(IV) complex containing bulky hydrophobic groups and NH···S hydrogen bonds, (Et4N)[Mo(IV)(OSi(t)BuPh2)(1,2-S2-3,6-{(4-(t)BuC6H4)3CCONH}2C6H2)2], was synthesized. This complex promotes the oxygen-atom-transfer (OAT) reaction of DMSO by efficient uptake of the substrate into the active center. The clean OAT reaction of Me3NO is also achieved.

  17. Viscosity-Reducing Bulky-Salt Excipients Prevent Gelation of Protein, but Not Carbohydrate, Solutions.

    PubMed

    Kumar, Awanish; Klibanov, Alexander M

    2017-08-01

    The problem of gelation of concentrated protein solutions, which poses challenges for both downstream protein processing and liquid formulations of pharmaceutical proteins, is addressed herein by employing previously discovered viscosity-lowering bulky salts. Procainamide-HCl and the salt of camphor-10-sulfonic acid with L-arginine (CSA-Arg) greatly retard gelation upon heating and subsequent cooling of the model proteins gelatin and casein in water: Whereas in the absence of additives the proteins form aqueous gels within several hours at room temperature, procainamide-HCl for both proteins and also CSA-Arg for casein prevent gel formation for months under the same conditions. The inhibition of gelation by CSA-Arg stems exclusively from the CSA moiety: CSA-Na was as effective as CSA-Arg, while Arg-HCl was marginally or not effective. The tested bulky salts did not inhibit (and indeed accelerated) temperature-induced gel formation in aqueous solutions of all examined carbohydrates-starch, agarose, alginate, gellan gum, and carrageenan.

  18. Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

    PubMed Central

    Kong, Xu-Dong; Yuan, Shuguang; Li, Lin; Chen, She; Xu, Jian-He; Zhou, Jiahai

    2014-01-01

    Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates. PMID:25331869

  19. para-C-H Borylation of Benzene Derivatives by a Bulky Iridium Catalyst.

    PubMed

    Saito, Yutaro; Segawa, Yasutomo; Itami, Kenichiro

    2015-04-22

    A highly para-selective aromatic C-H borylation has been accomplished. By a new iridium catalyst bearing a bulky diphosphine ligand, Xyl-MeO-BIPHEP, the C-H borylation of monosubstituted benzenes can be affected with para-selectivity up to 91%. This catalytic system is quite different from the usual iridium catalysts that cannot distinguish meta- and para-C-H bonds of monosubstituted benzene derivatives, resulting in the preferred formation of meta-products. The para-selectivity increases with increasing bulkiness of the substituent on the arene, indicating that the regioselectivity of the present reaction is primarily controlled by steric repulsion between substrate and catalyst. Caramiphen, an anticholinergic drug used in the treatment of Parkinson's disease, was converted into five derivatives via our para-selective borylation. The present [Ir(cod)OH]2/Xyl-MeO-BIPHEP catalyst represents a unique, sterically controlled, para-selective, aromatic C-H borylation system that should find use in streamlined, predictable chemical synthesis and in the rapid discovery and optimization of pharmaceuticals and materials.

  20. Structural phase transitions and adduct release in calcium borohydride

    SciTech Connect

    Paolone, A.; Palumbo, O.; Rispoli, P.; Miriametro, A.; Cantelli, R.; Luedtke, A.; Rönnebro, E.; Chandra, D.

    2011-09-01

    Ca(BH4)2 compounds were investigated above room temperature by anelastic spectroscopy (AS) and concomitant measurements of thermogravimetry and mass spectrometry (TGA/MS). Both AS and TGA/MS indicate that even after a thermal treatment at 125 °C for 20 h, a non-negligible residual of THF adduct is still present in the sample, which can be removed on a subsequent thermal treatment at temperatures lower than 250 °C. Above 250 °C dehydrogenation takes place. Moreover, AS sensitively detects the occurrence of the α → α’ structural phase transition around 180 °C, and the α’ → β transformation, which is completed around 330 °C. Finally, we also show that both transitions are irreversible and are not accompanied by a latent heat.

  1. Cytochrome c Adducts with PCB Quinoid Metabolites

    PubMed Central

    Li, Miao; Teesch, Lynn M.; Murry, Daryl J.; Pope, R. Marshal; Li, Yalan; Robertson, Larry W.; Ludewig, Gabriele

    2015-01-01

    PCBs are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy- metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and thereby cause defects in the function of cytochrome c. In this study synthetic PCB quinones (2-(4’-chlorophenyl)-1,4-benzoquinone, 2-(3’, 5’-dichlorophenyl)-1,4-benzoquinone, 2-(3’,4’, 5’-trichlorophenyl)-1,4-benzoquinone, and 2-(4’-chlorophenyl)-3,6-dichloro-1,4-benzoquinone) were incubated with cytochrome c, and adducts were detected by LC-MS and MALDI TOF. SDS PAGE gel electrophoresis was employed to separate the adducted proteins, while trypsin digestion and LC-MS/MS were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-para-quinone was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS PAGE gel. Cytochrome c was found to be in the reduced form after incubation with PCB quinones. These data provide evidence that the covalent binding of PCB quinone metabolites to cytochrome c may be included among the toxic effects of PCBs. PMID:26062463

  2. Human DNA adduct measurements: State of the art

    SciTech Connect

    Poirier, M.C.; Weston, A.

    1996-10-01

    Human DNA adduct formation (covalent modification of DNA with chemical carcinogens) is a promising biomarker for elucidating the molecular epidemiology of cancer. Classes of compounds for which human DNA adducts have been observed include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, mycotoxins, aromatic amines, heterocyclic amines, ultraviolet light, and alkylating cancer chemotherapeutic agents. Most human DNA adduct exposure monitoring has been performed with either {sup 32}P-postlabeling or immunoassays, neither of which is able to chemically characterize specific DNA adducts. Recently developed combinations of methods with chemical and physical end points have allowed identification of specific adducts in human tissues. Studies are presented that demonstrate that high ambient levels of benzo[a]pyrene are associated with high levels of DNA adducts in human blood cell DNA and that the same DNA adduct levels drop when the ambient PAH levels decrease significantly. DNA adduct dosimetry, which has been achieved with some dietary carcinogens and cancer chemotherapeutic agents, is described, as well as studies correlating DNA adducts with other biomarkers. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects, including oxidative damage, and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. The studies presented here may serve as useful prototypes for exploration of other toxicological end points. 156 refs., 1 fig., 3 tabs.

  3. Immunochemical properties of malondialdehyde-protein adducts.

    PubMed

    Lung, C C; Fleisher, J H; Meinke, G; Pinnas, J L

    1990-03-27

    Malondialdehyde (MDA), a product of lipid peroxidation, can bind to and modify proteins by adduct formation. To determine whether MDA adducts were immunogenic, MDA was added to rabbit serum albumin (RSA) in order to characterize MDA-proteins and to immunize rabbits. Bound MDA was proportional to the concentration of MDA added in the range of 2.5-20 mM as measured by thiobarbituric acid reactivity. MDA adducts of RSA migrated further toward the anode than native serum protein in zone and immunoelectrophoresis indicating increased negative charge. Rabbits immunized with MDA-RSA produced high titers of IgG antibodies to MDA-RSA as measured by enzyme-linked immunosorbent assay (ELISA). Hapten specificity of the antibody was demonstrated by antisera reactivity with MDA-RSA but not with unaltered RSA. Our findings support the possibility that MDA may serve as a hapten to form neoantigens which may represent a pathway by which lipid peroxidation could produce tissue damage via an immunologic mechanism.

  4. Development of an immunoassay to detect benzene adducts in hemoglobin

    SciTech Connect

    Grassman, J.A.

    1993-01-01

    The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failed to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.

  5. The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance.

    PubMed

    Rawat, Richa; Whitty, Adrian; Tonge, Peter J

    2003-11-25

    Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K(-1) = 16 +/- 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall Ki = 0.75 +/- 0.08 nM, reconciles existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first order rate constant for conversion of the initial EI complex to EI* (k2 = 0.13 +/- 0.01 min(-1)) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, I21V, I47T, and S94A, have little impact on the inhibition constants. Thus, drug resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.

  6. The influence of isometric hip adduction on quadriceps femoris activity.

    PubMed

    Hodges, P W; Richardson, C A

    1993-06-01

    In the treatment of muscle imbalances around the knee, hip adduction and the contraction of adductor magnus have been used to facilitate vastus medialis obliquus (VMO) to a greater extent than vastus lateralis (VL). This study was conducted to investigate the effectiveness of this technique. Hip adduction was superimposed onto the contraction of quadriceps femoris in a weight bearing (WB) and a non-weight bearing (NWB) position at three levels of hip adduction force. The muscle activity of VMO and VL was recorded using electromyography with the ratio of the recordings of VMO to VL used for comparison. The activity of VMO compared with VL was greater in WB than NWB without hip adduction. VMO activity increased relatively more than VL with the addition of each level of hip adduction in WB and only with maximal hip adduction in NWB. The results provide support for the use of this technique.

  7. Infrared spectra of some acetone—magnesium adducts

    NASA Astrophysics Data System (ADS)

    Hisatsune, I. C.

    Co-deposition of atomic magnesium with excess acetone at liquid-nitrogen temperature produces an unstable charge-transfer complex with a characteristic carbonyl infrared band at 1595 cm -1 and stable acetone adducts in which the metal atom bridges the carbonyl bond (π-complex) or coordinates to the oxygen atom (σ-complex). Infrared spectra of these complexes with (CH 3) 2CO and (CD 3) 2CO have been obtained. Corroborations for these adducts were obtained from infrared studies of acetone matrices with atomic copper and acetaldehyde matrices with atomic magnesium and with atomic copper. Infrared spectra of an acetone adduct and a dimethyl ether adduct of the Grignard reagent CH 3MgI have also been obtained. Hydrolysis of a σ-adduct gives acetone but isopropyl alcohol is obtained from hydrolysis of the π-adduct.

  8. Trimethylsilylmethyl complexes of the rare-earth metals with sterically hindered N-heterocyclic carbene ligands: adduct formation and C-H bond activation.

    PubMed

    Fegler, Waldemar; Spaniol, Thomas P; Okuda, Jun

    2010-08-07

    Tris(trimethylsilylmethyl) complexes of yttrium and lutetium [LnR(3)(THF)(2)] (R = CH(2)SiMe(3)) were treated with sterically bulky N-heterocyclic carbenes (NHC) 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene (IPr) and 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (IMes). IPr gave labile mono-adducts [LnR(3)(NHC)], isolated as thermally robust crystals and fully characterized by NMR spectroscopy and X-ray diffraction. IMes gave a similar lutetium mono-adduct [LuR(3)(IMes)] with the lutetium alkyl [LuR(3)(THF)(2)], whereas the yttrium alkyl [YR(3)(THF)(2)] resulted in the formation of an ortho-metalated product. This compound, isolated as a crystalline bis(THF) adduct, contains a strained six-membered chelate ring that has been formed by the C-H bond activation of one of the ortho-methyl groups of the mesityl group. In contrast [LuR(3)(IMes)] only slowly underwent a similar C-H bond activation.

  9. Protein modification by acrolein: Formation and stability of cysteine adducts

    PubMed Central

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2010-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  10. Role of malondialdehyde-acetaldehyde adducts in liver injury.

    PubMed

    Tuma, Dean J

    2002-02-15

    Malondialdehyde and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. MAA-protein adducts are composed of two major products whose structures and mechanism of formation have been elucidated. MAA adduct formation, especially in the liver, has been demonstrated in vivo during ethanol consumption. These protein adducts are capable of inducing a potent immune response, resulting in the generation of antibodies against both MAA epitopes, as well as against epitopes on the carrier protein. Chronic ethanol administration to rats results in significant circulating antibody titers against MAA-adducted proteins, and high anti-MAA titers have been associated with the severity of liver damage in humans with alcoholic liver disease. In vitro exposure of liver endothelial or hepatic stellate cells to MAA adducts induces a proinflammatory and profibrogenic response in these cells. Thus, during excessive ethanol consumption, ethanol oxidation and ethanol-induced oxidative stress result in the formation of acetaldehyde and malondialdehyde, respectively. These aldehydes can react together synergistically with proteins and generate MAA adducts, which are very immunogenic and possess proinflammatory and profibrogenic properties. By virtue of these potentially toxic effects, MAA adducts may play an important role in the pathogenesis of alcoholic liver injury.

  11. What predicts the first peak of the knee adduction moment?

    PubMed Central

    Schmitz, Anne; Noehren, Brian

    2014-01-01

    Introduction The first peak of the knee adduction moment curve during walking has been shown to be a good clinical surrogate measure of medial tibiofemoral joint loading and osteoarthritis. Defining the relative contributions of the variables that dictate the knee adduction moment, such as center of mass, center of pressure, vertical ground reaction force, and knee adduction angle (i.e. lower limb alignment), has not been formally investigated within the same cohort of individuals. Purpose Therefore, the goal of this study was to determine which of these variables is the biggest determinant of the first peak of knee adduction moment curve. Methods Instrumented gait analysis was collected for 30 individuals. Variables significantly correlated with the peak knee adduction moment were input into a stepwise multi-variable linear regression model. Results The knee adduction angle predicted 58% of the variance in the first peak knee adduction moment and the vertical ground reaction force magnitude predicted the second most variance (20%). Conclusions The most effective way to modify the peak knee adduction moment may be to change the knee adduction angle (e.g. offloader brace), followed by changing the vertical magnitude of the ground reaction force (e.g. cane use). PMID:25127390

  12. Electrospray ionization mass spectrometric characterization of acrylamide adducts to hemoglobin

    SciTech Connect

    Springer, D.L.; Goheen, S.C.; Edmonds, C.G. ); Bull, R.J.; Sylvester, D.M. )

    1993-01-01

    The most common procedure to identify hemoglobin adducts has been to cleave the adducts from the protein and characterize the adducting species, by, for example, derivatization and gas chromatography/mass spectrometry. To extend these approaches we used electrospray ionization mass spectrometry (ESI-MS) to characterize adducted hemoglobin. For this we incubated [[sup 14]C]acrylamide with the purified human hemoglobin (type A[sub 0]) under conditions that yielded high adduct levels. When the hemoglobin was separated by reversed-phase high-performance liquid chromatography (HPLC), 65% of the radioactivity copurified with the [beta]-subunit. Three adducted species were prominent in the ESI mass spectrum of the intact [beta]-subunit, indicating acrylamide adduction (i.e., mass increase of 71 Da) and two addition unidentified moieties with mass increments of 102 and 135 Da. Endoproteinase Glu-C digestion of the adducted [beta]-subunit resulted in a peptide mixture that, upon reversed-phase HPLC separation, provided several radiolabeled peptides. Using ESI-MS we identified these as the V[sub 91-101] and V[sub 102-122] peptides that represent the cysteine-containing peptides of the [beta]-subunit. These results provide definitive information on acrylamide-modified human hemoglobin and demonstrate that ESI-MS provides valuable structure information on chemically adducted proteins. 30 refs., 9 figs., 3 tabs.

  13. Quantitation of carcinogen bound protein adducts by fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Gan, Liang-Shang; Otteson, Michael S.; Doxtader, Mark M.; Skipper, Paul L.; Dasari, Ramachandra R.; Tannenbaum, Steven R.

    1989-01-01

    A highly significant correlation of aflatoxin B 1 serum albumin adduct level with daily aflatoxin B 1 intake was observed in a molecular epidemiological study of aflatoxin carcinogenesis which used conventional fluorescence spectroscopy methods for adduct quantitation. Synchronous fluorescence spectroscopy and laser induced fluorescence techniques have been employed to quantitate antibenzo[ a]pyrene diol epoxide derived globin peptide adducts. Fast and efficient methods to isolate the peptide adducts as well as eliminate protein fluorescence background are described. A detection limit of several femtomoles has been achieved. Experimental and technical considerations of low temperature synchronous fluorescence spectroscopy and fluorescence line narrowing to improve the detection sensitivities are also presented.

  14. Specimen histology after one or two preoperative CF-252 implants for bulky stage IB cervical cancer

    SciTech Connect

    Maruyama, Y.; van Nagell, J.R.; Feola, J.M.; Beach, J.L.; Yoneda, J.; Donaldson, E.; Gallion, H.; Rowley, K.; Powell, D.

    1987-10-01

    Using hysterectomy specimens obtained 1 month after Cf-252 neutron brachytherapy plus fractionated radiotherapy, we determined the fraction of positive and negative specimens with neutron dose for bulky Stage IB cervical cancers. The specimens obtained and studied after an initial Cf-252 insertion when the sources were newer and less decayed were more frequently negative for histological evidence of cancer than after the sources had decayed and 2 insertions were needed. After two insertions to deliver a therapeutic dose preoperatively the specimens were more frequently positive. When a larger initial dose was delivered to the tumor a larger proportion of negative specimens was noted. The size of neutron dose fraction was important to local tumor clearance and to rendering the specimens negative as well as schedule in use.

  15. Tumor regression and histologic clearance after neutron brachytherapy for bulky localized cervical carcinoma

    SciTech Connect

    Maruyama, Y.; Yoneda, J.; Van Nagell, J.R.; Donaldson, E.S.; Hanson, M.; Powell, D.; Muir, W.

    1982-12-15

    The response of bulky, advanced Stage 1B and early Stage II carcinoma of the cervix to neutron brachytherapy (NT and radiotherapy) was studied using combined NT radiation and extrafascial hysterectomy with histologic evaluation. Scheduling of neutron therapy relative to external beam photon therapy, tumor volume, tumor stage, tumor histology, and clinical tumor clearance were assessed in these studied. NT was easily combined with surgery in this study. Low stage tumors, small tumor volume and early neutron implants (scheduled within +/- one week of the start of fractionated radiation) showed more frequent histologic clearance of tumor. Long-term tumor control has been achieved and failures developed distance metastases without pelvic or local recurrence. This experience indicates that NT was effective for tumor clearance and control and represents and promising new modality for localized, advanced tumor therapy.

  16. Tumor regression and histologic clearance after neutron brachytherapy for bulky localized cervical carcinoma

    SciTech Connect

    Maruyama, Y.; Yoneda, J.; Van Nagell, J.R.; Donaldson, E.S.; Hanson, M.; Powell, D.; Muir, W.

    1982-12-15

    The response of bulky, advanced Stage IB and early Stage II carcinoma of the cervix to neutron brachytherapy (NT and radiotherapy) was studied using combined NT radiation) and extrafascial hysterectomy with histologic evaluation. Scheduling of neutron therapy relative to external beam photon therapy, tumor volume, tumor stage, tumor histology, and clinical tumor clearance were assessed in these studies. NT was easily combined with surgery in this study. Low stage tumors, small tumor volume and ''early'' neutron implants (scheduled within +/- one week of the start of fractionated radiation) showed more frequent histologic clearance of tumor. Long-term tumor control has been achieved and failures developed distant metastases without pelvic or local recurrence. This experience indicates that NT was effective for tumor clearance and control and represents a promising new modality for localized, advanced tumor therapy.

  17. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines

    NASA Astrophysics Data System (ADS)

    Pavlidis, Ioannis V.; Weiß, Martin S.; Genz, Maika; Spurr, Paul; Hanlon, Steven P.; Wirz, Beat; Iding, Hans; Bornscheuer, Uwe T.

    2016-11-01

    The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

  18. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines.

    PubMed

    Pavlidis, Ioannis V; Weiß, Martin S; Genz, Maika; Spurr, Paul; Hanlon, Steven P; Wirz, Beat; Iding, Hans; Bornscheuer, Uwe T

    2016-11-01

    The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

  19. Mesostructured fatty acid-tethered silicas: sustaining the order by co-templating with bulky precursors.

    PubMed

    El Kadib, Abdelkrim; Katir, Nadia; Finiels, Annie; Castel, Annie; Marcotte, Nathalie; Molvinger, Karine; Biolley, Christine; Gaveau, Philippe; Bousmina, Mosto; Brunel, Daniel

    2013-02-07

    The co-condensation of functional alkoxysilanes with tetraethoxysilane in the presence of a structure directing agent under sol-gel process chemistry is a common way to access functional organosilica with an ordered mesostructure. In this report, bulky silylated fatty acid methyl esters were used both as co-templating bio-molecules and functionalizing agents in the process of supra-molecular silica mineralization. The highest structural regularity in terms of pore size distribution and channel size homogeneity was observed for carboxy-tethered silica possessing SBA-15-type architecture due to an enhanced fatty acid precursor-surfactant interaction. The carboxylic surface embedded within the hydrophobic environment of the fatty compounds confers to these materials interesting reactive-surface properties with promising applications as drug-delivery systems and bio-catalytic nanoreactors.

  20. Regioregular Phthalocyanines Substituted with Bulky Donors at Non-Peripheral Positions.

    PubMed

    Yamamoto, Satoshi; Kuribayashi, Kengo; Murakami, Takuro N; Kwon, Eunsang; Stillman, Martin J; Kobayashi, Nagao; Segawa, Hiroshi; Kimura, Mutsumi

    2017-09-08

    Three regioregular phthalocyanines (1-3) were synthesized selectively by the cyclic tetramerization of phthalonitriles bearing a bulky diarylamine substituent at the next position of nitrile. The steric repulsion at the tetramerization of bulky phthalonitriles allowed for the selective formation of regioregular phthalocyanines as confirmed by NMR and single crystal X-ray structural analyses. The absorption spectrum of 1 substituted with di(4-tert-buthylphenyl)amine groups at the non-peripheral positions showed a non-split Q band at 764 nm, which was red-shifted by 83 nm compared with that of metal free phthalocyanine (H2Pc). The TD-DFT calculation and electrochemical studies prove that the substitution of diarylamine groups at the α positions effectively destabilizes the HOMO energy level, which causes a large red-shift of the Q band. Moreover, 1 can generate a one-electron oxidation specie through chemical oxidation. The Q band position of 2 bearing 4,4'-dimetoxyphenylamine units was further shifted by 10 nm compared with that of 1. In addition, 3 having carbazole units showed a small red-shift of the Q band relative to H2Pc. The hole-mobility of 2 in thin film was determined to be 1.1 × 10-5 cm V-1 s-1 by using a space charge limited current method. A perovskite solar cell employing 2 as a hole-transporting layer gave an efficiency of 5.1 % under standard global 100 W cm-2 AM 1.5 G illumination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

    PubMed Central

    Sharma, Vaneet K; Glick, James; Liao, Qing; Shen, Chang; Vouros, Paul

    2012-01-01

    The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N-acetylaminofluorene (AAF) adducted 17-mer (5′OH-CCT ACC CCT TCC TTG TA-3′OH) oligonucleotide. Further computational screening of this AAF adducted 17-mer oligonucleotide (5′OH-CCT ACC CCT TCC TTG TA-3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15-mer oligonucleotide (5′OH-ATGAACCGGAGGCCC-3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. PMID:22689626

  2. The effect of orthotic devices on knee adduction moment, pain and function in medial compartment knee osteoarthritis: a literature review.

    PubMed

    Baghaei Roodsari, Roshanak; Esteki, Ali; Aminian, Gholamreza; Ebrahimi, Ismaeil; Mousavi, Mohammad Ebramim; Majdoleslami, Basir; Bahramian, Fatemeh

    2016-03-15

    Background Knee braces and foot orthoses are commonly used to improve knee adduction moment, pain and function in subjects with knee osteoarthritis (OA). However, no literature review has been performed to compare the effects of foot orthoses and knee braces in this group of patients. Purpose The aim of this review was to evaluate the effects of foot orthoses and knee braces on knee adduction moment, pain and function in individuals with knee OA. Study design Literature review. Method The search strategy was based on the Population Intervention Comparison Outcome method. A search was performed in PubMed, Science Direct, Google Scholar and ISI web of knowledge databases using the PRISMA method and based on selected keywords. Thirty-one related articles were selected for final evaluation. Results The results of the analysis of these studies demonstrated that orthotic devices reduce knee adduction moment and also improve pain and function in individuals with knee OA. Conclusion Foot orthoses may be more effective in improving pain and function in subjects with knee OA. Both knee braces and foot orthoses reduce the knee adduction moment in knee OA and consequently patients typically do not need to use knee braces for a long period of time. Also, foot orthoses and knee braces may be more effective for medial compartment knee OA patients due to the fact that this treatment helps improve pain and function. Implications for Rehabilitation Knee braces and foot orthoses are commonly used for improving knee adduction moment, pain and function in subjects with knee osteoarthritis (OA). Orthotic devices can reduce knee adduction moment, pain and improve function in knee OA. The combined use of a knee braces and foot orthoses can provide more improvement in knee adduction moment, reduced pain and increased function.

  3. Paracetamol (acetaminophen) protein adduct concentrations during therapeutic dosing.

    PubMed

    Heard, Kennon; Green, Jody L; Anderson, Victoria; Bucher-Bartelson, Becki; Dart, Richard C

    2016-03-01

    Paracetamol protein adducts (PPA) are a biomarker of paracetamol exposure. PPA are quantified as paracetamol-cysteine (APAP-CYS), and concentrations above 1.1 μmol l(-1) have been suggested as a marker of paracetamol-induced hepatotoxicity. However, there is little information on the range of concentrations observed during prolonged therapeutic dosing. The aim of the present study was to describe the concentration of PPA in the serum of subjects taking therapeutic doses of paracetamol for at least 16 days. Preplanned secondary aim of a prospective randomized controlled (placebo vs. 4g day(-1) paracetamol) trial. We measured subjects' serum PPA concentrations every 3 days for a minimum of 16 days. We also measured concentrations on study days 1-3 and 16-25 in subsets of patients. PPA were quantified as APAP-CYS after gel filtration and protein digestion using liquid chromatography/mass spectrometry. Ninety per cent of subjects had detectable PPA after five doses. Median APAP-CYS concentrations in paracetamol-treated subjects increased to a plateau of 0.1 μmol l(-1) on day 7, where they remained. The highest concentration measured was 1.1 μmol l(-1) and two subjects never had detectable PPA levels. PPA were detected in the serum of 78% of subjects 9 days after their final dose. PPA are detectable in the vast majority of subjects taking therapeutic doses of paracetamol. While most have concentrations well below the threshold associated with hepatotoxicity, concentrations may approach 1.1 μmol l(-1) in rare cases. Adducts are detectable after a few doses and can persist for over a week after dosing is stopped. © 2015 The British Pharmacological Society.

  4. Photo-cross-linking of XPC-Rad23B to cisplatin-damaged DNA reveals contacts with both strands of the DNA duplex and spans the DNA adduct.

    PubMed

    Neher, Tracy M; Rechkunova, Nadejda I; Lavrik, Olga I; Turchi, John J

    2010-02-02

    Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The xeroderma pigmentosum group C (XPC)-Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photo-cross-linking experiments revealed that the human XPC-Rad23B complex makes direct contact with both the cisplatin-damaged DNA strand and the complementary undamaged strand of a duplex DNA substrate. Coupling photo-cross-linking with denaturation and immunoprecipitation of protein-DNA complexes, we identified the XPC subunit in complex with damaged DNA. While the interaction of the XPC subunit with DNA was direct, studies revealed that although Rad23B was found in complex with DNA, the Rad23B-DNA interaction was largely indirect via its interaction with XPC. Using site specific cross-linking, we determined that the XPC-Rad23B complex is preferentially cross-linked to the damaged DNA when the photoreactive FAP-dCMP (exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine 5'-monophosphate) analogue is located to the 5' side of the cisplatin-DNA adduct. When the FAP-dCMP analogue is located to the 3' side of the adduct, no difference in binding was detected between undamaged and damaged DNA. Collectively, these data suggest a model in which XPC-DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential cross-linking 5' of the cisplatin-damaged site suggests that the XPC-Rad23B complex displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage.

  5. The antimicrobial activities of the cinnamaldehyde adducts with amino acids.

    PubMed

    Wei, Qing-Yi; Xiong, Jia-Jun; Jiang, Hong; Zhang, Chao; Wen Ye

    2011-11-01

    Cinnamaldehyde is a well-established natural antimicrobial compound. It is probable for cinnamaldehyde to react with amino acid forming Schiff base adduct in real food system. In this paper, 9 such kind of adducts were prepared by the direct reaction of amino acids with cinnamaldehyde at room temperature. Their antimicrobial activities against Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were evaluated with benzoic acid as a reference. The adducts showed a dose-dependent activities against the three microbial strains. Both cinnamaldehyde and their adducts were more active against B. subtilis than on E. coli, and their antimicrobial activities were higher at lower pH. Both cinnamaldehyde and its adducts were more active than benzoic acid at the same conditions. The adduct compound A was non-toxic by primary oral acute toxicity study in mice. However, in situ effect of the adduct compound A against E. coli was a little lower than cinnamaldehyde in fish meat. This paper for the first time showed that the cinnamaldehyde adducts with amino acids had similar strong antimicrobial activities as cinnamaldehyde, which may provide alternatives to cinnamaldehyde in food to avoid the strong unacceptable odor of cinnamaldehyde. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Monitoring the apple polyphenol oxidase-modulated adduct formation of phenolic and amino compounds.

    PubMed

    Reinkensmeier, Annika; Steinbrenner, Katrin; Homann, Thomas; Bußler, Sara; Rohn, Sascha; Rawel, Hashadrai M

    2016-03-01

    Minimally processed fruit products such as smoothies are increasingly coming into demand. However, they are often combined with dairy ingredients. In this combination, phenolic compounds, polyphenoloxidases, and amino compounds could interact. In this work, a model approach is presented where apple serves as a source for a high polyphenoloxidase activity for modulating the reactions. The polyphenoloxidase activity ranged from 128 to 333nakt/mL in different apple varieties. From these, 'Braeburn' was found to provide the highest enzymatic activity. The formation and stability of resulting chromogenic conjugates was investigated. The results show that such adducts are not stable and possible degradation mechanisms leading to follow-up products formed are proposed. Finally, apple extracts were used to modify proteins and their functional properties characterized. There were retaining antioxidant properties inherent to phenolic compounds after adduct formation. Consequently, such interactions may also be utilized to improve the textural quality of food products.

  7. Adduct Formation in ESI/MS by Mobile Phase Additives

    NASA Astrophysics Data System (ADS)

    Kruve, Anneli; Kaupmees, Karl

    2017-03-01

    Adduct formation is a common ionization method in electrospray ionization mass spectrometry (ESI/MS). However, this process is poorly understood and complicated to control. We demonstrate possibilities to control adduct formation via mobile phase additives in ESI positive mode for 17 oxygen and nitrogen bases. Mobile phase additives were found to be a very effective measure for manipulating the formation efficiencies of adducts. An appropriate choice of additive may increase sensitivity by up to three orders of magnitude. In general, sodium adduct [M + Na]+ and protonated molecule [M + H]+ formation efficiencies were found to be in good correlation; however, the former were significantly more influenced by mobile phase properties. Although the highest formation efficiencies for both species were observed in water/acetonitrile mixtures not containing additives, the repeatability of the formation efficiencies was found to be improved by additives. It is concluded that mobile phase additives are powerful, yet not limiting factors, for altering adduct formation.

  8. Adduct Formation in ESI/MS by Mobile Phase Additives

    NASA Astrophysics Data System (ADS)

    Kruve, Anneli; Kaupmees, Karl

    2017-05-01

    Adduct formation is a common ionization method in electrospray ionization mass spectrometry (ESI/MS). However, this process is poorly understood and complicated to control. We demonstrate possibilities to control adduct formation via mobile phase additives in ESI positive mode for 17 oxygen and nitrogen bases. Mobile phase additives were found to be a very effective measure for manipulating the formation efficiencies of adducts. An appropriate choice of additive may increase sensitivity by up to three orders of magnitude. In general, sodium adduct [M + Na]+ and protonated molecule [M + H]+ formation efficiencies were found to be in good correlation; however, the former were significantly more influenced by mobile phase properties. Although the highest formation efficiencies for both species were observed in water/acetonitrile mixtures not containing additives, the repeatability of the formation efficiencies was found to be improved by additives. It is concluded that mobile phase additives are powerful, yet not limiting factors, for altering adduct formation.

  9. [Testicular seminoma in stages I and II non-bulky. 16 years' experience].

    PubMed

    Maranzano, E; Latini, P; Leggio, M; Aristei, C; Panizza, B M; Perrucci, E; Lupattelli, M

    1994-06-01

    From June 1977 through June 1993, ninety-five patients with testicular seminoma were treated in our center. This paper reports on 67 assessable patients--52 with stage I and 15 with non-bulky stage II disease. Median follow-up is 8 years (range: 4-16 years). Postorchiectomy radiotherapy consisted in 30 Gy (1.5 Gy/day) precautionary treatment to ipsilateral hemipelvis and paraaortic nodes (stage I) or 40-45 Gy to the same area plus 25.5-30 Gy prophylactic irradiation to mediastinum and supraclavicular fossae (stage II). Ten-year actuarial survival is 100%-96.8% +/- 2.2 considering deaths from other diseases. Ten-year disease-free survival is 95.3% +/- 2.6. The 3 relapsed patients were rescued with chemotherapy or radiotherapy (1 and 2 cases, respectively). Acute side-effects were nausea (30% of cases) and vomiting (18%) which disappeared after oral antiemetics. Late toxicity-asymptomatic osteolysis of the ipsilateral pubic region--was observed in 1 patient only (1.5%) who received cobalt therapy to inguinal canal and hemiscrotum (40.5 Gy in 27 fractions). The current diagnostic and therapeutic approaches to testicular seminoma are discussed. In stage I the conventional treatment is low-dose (20-25 Gy) subdiaphragmatic radiotherapy and a policy of surveillance is justified only for clinical trials. In non-bulky stage II disease lumboaortic and hemipelvic irradiation (36-40 Gy) is the treatment of choice whereas precautionary irradiation should not be given to the mediastinum. If abdominal CT scans show nodal metastases, chest CT is necessary for staging instead of chest X-ray films. When abdominal CT findings are negative or questionable, bi-pedal lymphography must be performed. Residual testis US should be the routine examination for the early diagnosis of metachronous contralateral seminoma. The semen should be tested for further storage and sexual functions should be accurately analyzed to distinguish between organic and psychologic causes. Although limited, our

  10. The Formation and Biological Significance of N7-Guanine Adducts

    PubMed Central

    Boysen, Gunnar; Pachkowski, Brian F.; Nakamura, Jun; Swenberg, James A

    2009-01-01

    DNA alkylation or adduct formation occurs at nucleophilic sites in DNA, mainly the N7-position of guanine. Ever since identification of the first N7-guanine adduct, several hundred studies on DNA adducts have been reported. Major issues addressed include the relationships between N7-guanine adducts and exposure, mutagenesis, and other biological endpoints. It became quickly apparent that N7-guanine adducts are frequently formed, but may have minimal biological relevance, since they are chemically unstable and do not participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to be excellent biomarkers for internal exposure to direct acting and metabolically activated carcinogens. Questions arise, however, regarding the biological significance for N7-guanine adducts that are readily formed, do not persist, and are not likely to be mutagenic. Thus, we set out to review the current literature to evaluate their formation and the mechanistic evidence for the involvement of N7-guanine adducts in mutagenesis or other biological processes. It was concluded that there is insufficient evidence that N7-guanine adducts can be used beyond confirmation of exposure to the target tissue and demonstration of the molecular dose. There is little to no evidence that N7-guanine adducts or their depurination product, apurinic sites, are the cause of mutations in cells and tissues, since increases in AP sites have not been shown unless toxicity is extant. However, more research is needed to define the extent of chemical depurination versus removal by DNA repair proteins. Interestingly, N7-guanine adducts are clearly present as endogenous background adducts and the endogenous background amounts appear to increase with age. Furthermore, the N7-guanine adducts have been shown to convert to ring opened lesions (FAPy), which are much more persistent and have higher mutagenic potency. Studies in humans are limited in sample size and differences between controls and

  11. Application of a gas-solid fluidized bed separator for shredded municipal bulky solid waste separation.

    PubMed

    Sekito, T; Matsuto, T; Tanaka, N

    2006-01-01

    A laboratory-scale gas-solid fluidized bed separator able to separate fractions of 5.6-50mm was used for separation of shredded municipal bulky waste (SBW) into combustibles and incombustibles. In batch-scale tests, it was found that accumulation of SBW in the bottom of the bed significantly reduced the separation efficiency. In this study, stirring was shown to be effective in preventing this accumulation. Flexible sheet materials such as paper and film plastics also significantly decreased the separation efficiency. In batch-scale tests, an overall efficiency of 90% was obtained when flexible materials such as film plastics and paper were excluded from the feed SBW. In continuous feeding tests, purities of the float and sink fractions attained 95% and 86% efficiencies, respectively, with an overall efficiency of 79%. The effect of feedstock shape on separation efficiency was also investigated. This study revealed that large particles can be properly separated on the basis of density, while the shape of the material significantly influenced behavior in the fluidizing bed.

  12. Effect of Bulky Substituents in the Polymer Backbone on the Properties of Polyimide Aerogels.

    PubMed

    Viggiano, Rocco P; Williams, Jarrod C; Schiraldi, David A; Meador, Mary Ann B

    2017-03-08

    With unique advantages over inorganic aerogels including higher strengths and compressive moduli, greater toughness, and the ability to be fabricated as a flexible thin film, polymer aerogels have the potential to supplant inorganic aerogels in numerous applications. Among polymer aerogels, polyimide aerogels possess a high degree of high thermal stability as well as outstanding mechanical properties. However, while the onset of thermal decomposition for these materials is typically very high (greater than 500 °C), the polyimide aerogels undergo dramatic thermally induced shrinkage at temperatures well below their glass transition (Tg) or decomposition temperature, which limits their use. In this study, we show that shrinkage is reduced when a bulky moiety is incorporated in the polymer backbone. Twenty different formulations of polyimide aerogels were synthesized from 3,3,'4,4'-biphenyltetracarboxylic dianhydride (BPDA) and 4,4'-oxidianiline (ODA) or a combination of ODA and 9,9'-bis(4-aminophenyl)fluorene (BAPF) and cross-linked with 1,3,5-benzenetricarbonyl trichloride (BTC) in a statistically designed study. The polymer concentration, n-value, and molar concentration of ODA and BAPF were varied to demonstrate the effect of these variables on certain properties. Samples containing BAPF showed a reduction in shrinkage by as much as 50% after aging at elevated temperatures for 500 h compared to those made with ODA alone.

  13. Bulky “Gatekeeper” Residue Changes the Cosubstrate Specificity of Aminoglycoside 2″-Phosphotransferase IIa

    PubMed Central

    Bhattacharya, Monolekha; Toth, Marta; Smith, Clyde A.

    2013-01-01

    The aminoglycoside 2″-phosphotransferases APH(2″)-IIa and APH(2″)-IVa can utilize ATP and GTP as cosubstrates, since both enzymes possess overlapping but discrete structural templates for ATP and GTP binding. APH(2″)-IIIa uses GTP exclusively, because its ATP-binding template is blocked by a bulky tyrosine “gatekeeper” residue. Replacement of the “gatekeeper” residues M85 and F95 in APH(2″)-IIa and APH(2″)-IVa, respectively, by tyrosine does not significantly change the antibiotic susceptibility profiles produced by the enzymes. In APH(2″)-IIa, M85Y substitution results in an ∼10-fold decrease in the Km value of GTP and an ∼320-fold increase in the Km value of ATP. In APH(2″)-IVa, F95Y substitution results in a modest decrease in the Km values of both GTP and ATP. Structural analysis indicates that in the APH(2″)-IIa M85Y mutant, tyrosine blocks access of ATP to the correct position in the binding site, while the larger nucleoside triphosphate (NTP)-binding pocket of the APH(2″)-IVa F95Y mutant allows the tyrosine to move away, thus giving access to the ATP-binding template. PMID:23716051

  14. Bulky melamine-based Zn-porphyrin tweezer as a CD probe of molecular chirality.

    PubMed

    Petrovic, Ana G; Vantomme, Ghislaine; Negrón-Abril, Yashira L; Lubian, Elisa; Saielli, Giacomo; Menegazzo, Ileana; Cordero, Roselynn; Proni, Gloria; Nakanishi, Koji; Carofiglio, Tommaso; Berova, Nina

    2011-10-01

    The transfer of chirality from a guest molecule to an achiral host is the subject of significant interest especially when, upon chiral induction, the chiroptical response of the host/guest complex can effectively report the absolute configuration (AC) of the guest. For more than a decade, dimeric metalloporphyrin hosts (tweezers) have been successfully applied as chirality probes for determination of the AC for a wide variety of chiral synthetic compounds and natural products. The objective of this study is to investigate the utility of a new class of melamine-bridged Zn-porphyrin tweezers as sensitive AC reporters. A combined approach based on an experimental CD analysis and a theoretical prediction of the prevailing interporphyrin helicity demonstrates that these tweezers display favorable properties for chiral recognition. Herein, we discuss the application of the melamine-bridged tweezer to the chiral recognition of a diverse set of chiral guests, such as 1,2-diamines, α-amino-esters and amides, secondary alcohols, and 1,2-amino-alcohols. The bulky periphery and the presence of a rigid porphyrin linkage lead, in some cases, to a more enhanced CD sensitivity than that reported earlier with other tweezers.

  15. Catalytic deoxydehydration of diols to olefins by using a bulky cyclopentadiene-based trioxorhenium catalyst.

    PubMed

    Raju, Suresh; Jastrzebski, Johann T B H; Lutz, Martin; Klein Gebbink, Robertus J M

    2013-09-01

    A bulky cyclopentadienyl (Cp)-based trioxorhenium compound was developed for the catalytic deoxydehydration of vicinal diols to olefins. The 1,2,4-tri(tert-butyl)cyclopentadienyl trioxorhenium (2) catalyst was synthesised in a two-step synthesis procedure. Dirhenium decacarbonyl was converted into 1,2,4-tri(tert-butyl)cyclopentadienyl tricarbonyl rhenium, followed by a biphasic oxidation with H2 O2 . These two new three-legged compounds with a 'piano-stool' configuration were fully characterised, including their single crystal X-ray structures. Deoxydehydration reaction conditions were optimised by using 2 mol % loading of 2 for the conversion of 1,2-octanediol into 1-octene. Different phosphine-based and other, more conventional, reductants were tested in combination with 2. Under optimised conditions, a variety of vicinal diols (aromatic and aliphatic, internal and terminal) were converted into olefins in good to excellent yields, and with minimal olefin isomerisation. A high turnover number of 1400 per Re was achieved for the deoxydehydration of 1,2-octanediol. Furthermore, the biomass-derived polyols (glycerol and erythritol) were converted into their corresponding olefinic products by 2 as the catalyst.

  16. Theoretical evaluation of zeolite confinement effects on the reactivity of bulky intermediates.

    PubMed

    Lesthaeghe, David; Van Speybroeck, Veronique; Waroquier, Michel

    2009-07-14

    Zeolites provide a unique setting for heterogeneous Brønsted acid catalysis, because the effects of the surrounding framework on fundamental reaction kinetics go well beyond what would be expected for a mere reaction flask. This aspect becomes very pronounced when bulky molecules form key intermediates for the reaction under study, which is exactly when the interaction between the framework and the intermediate is maximal. We will use the example of methanol-to-olefin conversion (MTO), and, more specifically, the constant interplay between the inorganic host framework and the organic hydrocarbon pool co-catalyst, to illustrate how zeolite confinement directly influences catalytic reaction rates. Theoretical calculations are used to isolate and quantify these specific effects, with the main focus on methylbenzenes in ZSM-5, as the archetypical MTO catalyst. This review intends to give an overview of recent theoretical insights, which have proven to provide an ideal complementary tool to experimental investigations. In addition, we will also introduce the role of zeolite breathing in activating a catalytic cycle.

  17. Immunochemical quantitation of adducts induced in DNA by cis-diamminedichloroplatinum (II) and analysis of adduct-related DNA-unwinding.

    PubMed

    Fichtinger-Schepman, A M; Baan, R A; Luiten-Schuite, A; van Dijk, M; Lohman, P H

    1985-11-01

    Antibodies elicited against the haptens cis-Pt(NH3)2dGuodGMP and its ribo-analog, both covalently coupled to bovine serum albumin, recognize adducts of cis-diamminedichloroplatinum(II) (cis-DDP) in DNA. Antibody-binding to cis-DDP-DNA strongly depends on the accessibility of the adducts to the antibodies. In double-stranded cis-DDP-DNA with low Pt: nucleotide ratios (rb's), this accessibility is enhanced by unwinding of the cis-DDP-DNA, e.g. by heat-denaturation. An unwinding effect is also induced by the cis-DDP treatment itself. A260nm readings of cis-DDP-DNA samples indicate an increased denaturation of the DNA at increasing Pt-contents. The data obtained after heat-denaturation of the same samples show a growing capability to renaturation when the rb-values increase from 0 to 0.04; at 0.04 less than rb less than 0.18 the renaturation effect gradually disappears. In the competitive enzyme-linked immunosorbent assay (ELISA), the cis-DDP-adducts in heat-denatured DNA are detected in the pmol range; in DNA-digests, however, they are recognized in fmol amounts. For the individual Pt-containing (oligo)nucleotides the amounts causing 50% inhibition in the ELISA were established for the two anti-sera; they were 13.3 +/- 3.8 (fmol +/- S.D.) and 5.4 +/- 1.8 for cis-Pt(NH3)2d(GMP)2; 15.5 +/- 5.4 and 4.0 +/- 1.5 for cis-Pt(NH3)2d(pGpG); (2.6 +/- 1.1) X 10(3) and (2.0 +/- 1.0) X 10(3) for cis-Pt(NH3)2d(pApG); (5.6 +/- 1.9) X 10(3) and (2.9 +/- 0.4) X 10(3) for Pt(NH3)3dGMP. Pt-adducts in a trans-DDP-DNA digest are recognized in pmol amounts and dGMP in nmol quantitatives. Finally, the usefulness of these antibodies for the detection and quantitation of individual cis-DDP-adducts in cis-DDP-DNA digests was demonstrated.

  18. Biocatalytic Reductions of Baylis - Hillman Adducts

    SciTech Connect

    A Walton; W Conerly; Y Pompeu; B Sullivan; J Stewart

    2011-12-31

    Baylis-Hillman adducts are highly useful synthetic intermediates; to enhance their value further, we sought enantiocomplementary alkene reductases to introduce chirality. Two solutions emerged: (1) a wild-type protein from Pichia stipitis (OYE 2.6), whose performance significantly outstrips that of the standard enzyme (Saccharomyces pastorianus OYE1), and (2) a series of OYE1 mutants at position 116 (Trp in the wild-type enzyme). To understand how mutations could lead to inverted enantioselectivity, we solved the X-ray crystal structure of the Trp116Ile OYE1 variant complexed with a cyclopentenone substrate. This revealed key protein-ligand interactions that control the orientation of substrate binding above the FMN cofactor.

  19. DNA adduct formation by alachlor metabolites

    SciTech Connect

    Brown, M.A.; Kimmel, E.C.; Casida, J.E.

    1988-01-01

    The extent of DNA adduct formation by alachlor (ArN(CH/sub 2/OCH/sub 3/)C(O)CH/sub 2/Cl wherein Ar is 2,6-diethylphenyl) and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. (/sup 14/C-phenyl)Alachlor is compared to its two metabolic cleavage products, (/sup 14/C-phenyl) 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) (ArNHC(O)CH/sub 2/Cl) and (/sup 14/C-phenyl)2,6-diethylaniline (DEA) (ArNH/sub 2/), and to (/sup 14/C-methoxy)alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CEDPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from (/sup 14/C-methoxy)- than from (/sup 14/C-phenyl)alachlor. This 4-fold preferential DNA labeling from the /sup 14/C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with (/sup 14/C-phenyl)alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with (/sup 14/C-methoxy)alachlor.

  20. Glottal Adduction and Subglottal Pressure in Singing.

    PubMed

    Herbst, Christian T; Hess, Markus; Müller, Frank; Švec, Jan G; Sundberg, Johan

    2015-07-01

    Previous research suggests that independent variation of vocal loudness and glottal configuration (type and degree of vocal fold adduction) does not occur in untrained speech production. This study investigated whether these factors can be varied independently in trained singing and how subglottal pressure is related to average glottal airflow, voice source properties, and sound level under these conditions. A classically trained baritone produced sustained phonations on the endoscopic vowel [i:] at pitch D4 (approximately 294 Hz), exclusively varying either (a) vocal register; (b) phonation type (from "breathy" to "pressed" via cartilaginous adduction); or (c) vocal loudness, while keeping the others constant. Phonation was documented by simultaneous recording of videokymographic, electroglottographic, airflow and voice source data, and by percutaneous measurement of relative subglottal pressure. Register shifts were clearly marked in the electroglottographic wavegram display. Compared with chest register, falsetto was produced with greater pulse amplitude of the glottal flow, H1-H2, mean airflow, and with lower maximum flow declination rate (MFDR), subglottal pressure, and sound pressure. Shifts of phonation type (breathy/flow/neutral/pressed) induced comparable systematic changes. Increase of vocal loudness resulted in increased subglottal pressure, average flow, sound pressure, MFDR, glottal flow pulse amplitude, and H1-H2. When changing either vocal register or phonation type, subglottal pressure and mean airflow showed an inverse relationship, that is, variation of glottal flow resistance. The direct relation between subglottal pressure and airflow when varying only vocal loudness demonstrated independent control of vocal loudness and glottal configuration. Achieving such independent control of phonatory control parameters would be an important target in vocal pedagogy and in voice therapy. Copyright © 2015 The Voice Foundation. Published by Elsevier Inc

  1. Prolonged Acetaminophen-Protein Adduct Elimination During Renal Failure, Lack of Adduct Removal by Hemodiafiltration, and Urinary Adduct Concentrations After Acetaminophen Overdose.

    PubMed

    Curry, Steven C; Padilla-Jones, Angela; O'Connor, Ayrn D; Ruha, Anne-Michelle; Bikin, Dale S; Wilkins, Diana G; Rollins, Douglas E; Slawson, Matthew H; Gerkin, Richard D

    2015-06-01

    Elevated concentrations of serum acetaminophen-protein adducts, measured as protein-derived acetaminophen-cysteine (APAP-CYS), have been used to support a diagnosis of APAP-induced liver injury when histories and APAP levels are unhelpful. Adducts have been reported to undergo first-order elimination, with a terminal half-life of about 1.6 days. We wondered whether renal failure would affect APAP-CYS elimination half-life and whether continuous venovenous hemodiafiltration (CVVHDF), commonly used in liver failure patients, would remove adducts to lower their serum concentrations. Terminal elimination half-lives of serum APAP-CYS were compared between subjects with and without renal failure in a prospective cohort study of 168 adults who had ingested excessive doses of APAP. APAP-CYS concentrations were measured in plasma ultrafiltrate during CVVHDF at times of elevated serum adduct concentrations. Paired samples of urine and serum APAP-CYS concentrations were examined to help understand the potential importance of urinary elimination of serum adducts. APAP-CYS elimination half-life was longer in 15 renal failure subjects than in 28 subjects with normal renal function (41.3 ± 2.2 h versus 26.8 ± 1.1 h [mean ± SEM], respectively, p < 0.001). CVVHDF failed to remove detectable amounts of APAP-CYS in any of the nine subjects studied. Sixty-eight percent of 557 urine samples from 168 subjects contained no detectable APAP-CYS, despite levels in serum up to 16.99 μM. Terminal elimination half-life of serum APAP-CYS was prolonged in patients with renal failure for reasons unrelated to renal urinary adduct elimination, and consideration of prolonged elimination needs to be considered if attempting back-extrapolation of adduct concentrations. CVVHDF did not remove detectable APAP-CYS, suggesting approximate APAP-protein adduct molecular weights ≥ 50,000 Da. The presence of urinary APAP-CYS in the minority of instances was most compatible with renal

  2. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis.

    PubMed

    Agerbirk, Niels; De Nicola, Gina Rosalinda; Olsen, Carl Erik; Müller, Caroline; Iori, Renato

    2015-10-01

    Isothiocyanates form adducts with a multitude of biomolecules, and these adducts need analytical methods. Likewise, analytical methods for hydrophilic isothiocyanates are needed. We considered reaction with ammonia to form thiourea derivatives. The hydrophilic, glycosylated isothiocyanate moringin, 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate, was efficiently derivatized to the thiourea derivative by incubation with ammonia. The hydrophobic benzyl isothiocyanate was also efficiently derivatized to the thiourea derivative. The thiourea group provided a UV absorbing chromophore, and the derivatives showed expectable sodium and hydrogen adducts in ion trap mass spectrometry and were suitable for liquid chromatography analysis. Reactive dithiocarbamate adducts constitute the major type of reactive ITC adduct expected in biological matrices. Incubation of a model dithiocarbamate with ammonia likewise resulted in conversion to the corresponding thiourea derivative, suggesting that a variety of matrix-bound reactive isothiocyanate adducts can be determined using this strategy. As an example of the application of the method, recovery of moringin and benzyl isothiocyanate applied to cabbage leaf discs was studied in simulated insect feeding assays. The majority of moringin was recovered as native isothiocyanate, but a major part of benzyl isothiocyanate was converted to reactive adducts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Quantitation of DNA adducts by stable isotope dilution mass spectrometry

    PubMed Central

    Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

    2012-01-01

    Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

  4. Immunodetection of Serum Albumin Adducts as Biomarkers for Organophosphorus Exposure

    PubMed Central

    Chen, Sigeng; Zhang, Jun; Lumley, Lucille

    2013-01-01

    A major challenge in organophosphate (OP) research has been the identification and utilization of reliable biomarkers for the rapid, sensitive, and efficient detection of OP exposure. Although Tyr 411 OP adducts to human serum albumin (HSA) have been suggested to be one of the most robust biomarkers in the detection of OP exposure, the analysis of HSA-OP adduct detection has been limited to techniques using mass spectrometry. Herein, we describe the procurement of two monoclonal antibodies (mAb-HSA-GD and mAb-HSA-VX) that recognized the HSA Tyr 411 adduct of soman (GD) or S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate (VX), respectively, but did not recognize nonphosphonylated HSA. We showed that mAb-HSA-GD was able to detect the HSA Tyr 411 OP adduct at a low level (i.e., human blood plasma treated with 180 nM GD) that could not be detected by mass spectrometry. mAb-HSA-GD and mAb-HSA-VX showed an extremely low-level detection of GD adducted to HSA (on the order of picograms). mAb-HSA-GD could also detect serum albumin OP adducts in blood plasma samples from different animals administered GD, including rats, guinea pigs, and monkeys. The ability of the two antibodies to selectively recognize nerve agents adducted to serum albumin suggests that these antibodies could be used to identify biomarkers of OP exposure and provide a new biologic approach to detect OP exposure in animals. PMID:23192655

  5. Analytical methods in DNA and protein adduct analysis.

    PubMed

    Koivisto, Pertti; Peltonen, Kimmo

    2010-11-01

    DNA or protein adducts are reaction products of endogenous or exogenous chemicals and cellular macromolecules. Adducts are useful in toxicological studies and/or human biomonitoring exercises. In particular, DNA damage provides invaluable information for risk analysis. Second, metabolites or conjugates can be regarded as markers of phase II reactions though they may not give accurate information about the levels of reactive and damage-provoking reactive compounds or intermediates. Electrophiles are often short-lived molecules and therefore difficult to monitor. In contrast, adducts are often chemically stable, though their levels in biological samples are low, which makes their detection challenging. The assay of adducts is similar to the analysis of any other trace organic molecule, i.e. problems with the matrix and small amounts of analytes in samples. The (32)P-postlabelling assay is a specific method for DNA adducts but immunochemical and fluorescence-based methods have been developed which can detect adducts linked to both DNA and protein. Tandem mass spectrometry, particularly if combined with ultrahigh-performance liquid chromatography, is currently the recommended detection technique; however investigators are striving to develop novel ways to achieve greater sensitivity. Standards are a prerequisite in adduct analysis, but unfortunately they are seldom commercially available.

  6. The reversibility of the glutathionyl-quercetin adduct spreads oxidized quercetin-induced toxicity

    SciTech Connect

    Boots, Agnes W. . E-mail: a.boots@farmaco.unimaas.nl; Balk, Jiska M.; Bast, Aalt; Haenen, Guido R.M.M.

    2005-12-16

    Quercetin is one of the most prominent dietary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its o-quinone/quinone methide QQ. QQ is toxic since it instantaneously reacts with thiols of, e.g., proteins. In cells, QQ will initially form an adduct with glutathione (GSH), giving GSQ. We have found that GSQ is not stable; it dissociates continuously into GSH and QQ with a half life of 2 min. Surprisingly, GSQ incubated with 2-mercapto-ethanol (MSH), a far less reactive thiol, results in the conversion of GSQ into the MSH-adduct MSQ. A similar conversion of GSQ into relatively stable protein thiol-quercetin adducts is expected. With the dithiol dihydrolipoic acid (L(SH){sub 2}), quercetin is formed out of GSQ. These results indicate that GSQ acts as transport and storage of QQ. In that way, the initially highly focussed toxicity of QQ is dispersed by the formation of GSQ that finally spreads QQ-induced toxicity, probably even over cells.

  7. Cellulose based hybrid hydroxylated adducts for polyurethane foams

    NASA Astrophysics Data System (ADS)

    De Pisapia, Laura; Verdolotti, Letizia; Di Mauro, Eduardo; Di Maio, Ernesto; Lavorgna, Marino; Iannace, Salvatore

    2012-07-01

    Hybrid flexible polyurethane foams (HPU) were synthesized by using a hybrid hydroxilated adduct (HHA) based on renewable resources. In particular the HHA was obtained by dispersing cellulose wastes in colloidal silica at room temperature, pressure and humidity. The colloidal silica was selected for its ability of modifying the cellulose structure, by inducing a certain "destructurization" of the crystalline phase, in order to allow cellulose to react with di-isocyanate for the final synthesis of the polyurethane foam. In fact, cellulose-polysilicate complexes are engaged in the reaction with the isocyanate groups. This study provides evidence of the effects of the colloidal silica on the cellulose structure, namely, a reduction of the microfiber cellulose diameter and the formation of hydrogen bonds between the polysilicate functional groups and the hydroxyl groups of the cellulose, as assessed by IR spectroscopy and solid state NMR. The HHA was added to a conventional polyol in different percentages (between 5 and 20%) to synthesize HPU in presence of catalysts, silicone surfactant and diphenylmethane diisocyanate (MDI). The mixture was expanded in a mold and cured for two hours at room temperature. Thermal analysis, optical microscopy and mechanical tests were performed on the foams. The results highlighted an improvement of thermal stability and a decrease of the cell size with respect neat polyurethane foam. Mechanical tests showed an improvement of the elastic modulus and of the damping properties with increasing HHA amount.

  8. Impact of Hyperglycemia on Outcomes among Patients Receiving Neoadjuvant Chemotherapy for Bulky Early Stage Cervical Cancer

    PubMed Central

    Lu, Huai-wu; Zhang, Bing-zhong; Wang, Li-juan; Lin, Zhong-qiu

    2016-01-01

    Background The impact of hyperglycemia on survival of patients undergoing neoadjuvant chemotherapy (NACT) for bulky early stage cervical cancer (BESCC) has not been explored. Method Records of patients who received NACT and radical hysterectomy in our institution between January 2005 and June 2010 were reviewed. Results In total, 347 patients were included. The median follow-up time was 37 months (range: 4–65). Patients with hyperglycemia (fasting blood glucose ≥ 100 mg/dl) had shorter recurrence-free survival (RFS) (univariate hazard ratio [HR] = 1.95, 95% confidence interval [CI] [1.16, 3.28], P = 0.010) and cancer-specific survival (CSS) (univariate HR = 2.24, 95% CI [1.33, 3.78], P = 0.002) compared with those with euglycemia (fasting blood glucose <100 mg/dl). In multivariate analysis, positive surgical margins, parametrium invasion, node metastasis, hyperglycemia and complete response to NACT independently predicted recurrence and cancer-specific death. To further validate the prognostic value of hyperglycemia, we conducted a subgroup analysis based on patient baseline characteristics and prognostic effect of hyperglycemia remained significant in all subgroups. On multivariable logistic regression analysis, euglycemia before NACT, squamous cell tumor and pre-treatment squamous cell carcinoma antigen levels < 3.5 ng/ml were identified as independent predictors of complete response after NACT. Conclusions FBG ≥100 mg/dl is a negative prognostic predictor for cervical cancer patients receiving NACT for BESCC. Patients with hyperglycemia are less likely to achieve complete response after NACT. Our findings underscore the clinical utility of hyperglycemia screening of for cervical cancer patients. PMID:27851819

  9. Plaque pH Changes Following Consumption of Two Types of Plain and Bulky Bread

    PubMed Central

    Mortazavi, Shiva; Noin, Sogol

    2011-01-01

    Background: Consistency, backing process and content differences could influence cariogenic potential of foods. The aim was to compare plaque pH changes following consumption of two types of bread with different physical characteristics. Methods: In this clinical trial, interproximal plaque pH of 10 volunteers with high risk of dental caries (saliva Streptococcus mutans > 105, high dental caries experience, and average DMFT =6.10 ± 1.56) was measured. Plain traditionally backed “Sangak bread” and soft bulky “Baguette bread” and %10 sucrose solution were tested in a cross over designed experiment. Baseline plaque pH was recorded and followed by 1, 5, 10, 15, 20, and 30 minutes intervals. Data was analyzed using ANOVA and Tukey test (α = 0.05). Results: Sucrose solution caused the most pronounced pH and ΔpH drop from 7.15 ± 0.33 at baseline to 6.78 ± 0.29. Means plaque pH of 10% sucrose solution and Baguette were not statistically different at 1, 20 and 30 minutes (P > 0.05). Mean plaque pH of Sangak and Baguette showed significant differences at 0, 1, 20 and30 minutes (P <60; 0.05). Sucrose solution caused a dramatic plaque pH drop during first 10 minutes and then within 30 minutes returned to baseline pH. For two bread samples within first 10 minutes, pH increased and then started to decrease during tenth to fifteenth minutes. Conclusion: During all experiment phases, the mean pH of Baguette with less consistency and carbohydrate content and higher rate of starch gelatination was lower compared to Sangak. PMID:22013467

  10. A fluorescence-based analysis of aristolochic acid-derived DNA adducts.

    PubMed

    Romanov, Victor; Sidorenko, Victoria; Rosenquist, Thomas A; Whyard, Terry; Grollman, Arthur P

    2012-08-01

    Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.

  11. Concomitant use of radiotherapy and two topoisomerase inhibitors to treat adult T-cell leukemia with a radiotherapy-resistant bulky disease: a case series.

    PubMed

    Obama, Kosuke

    2014-01-01

    Concomitant chemoradiotherapy is established as the standard treatment to improve the prognosis of several types of solid tumor, but has not been the general practice for hematological malignancies. Here, I report two cases of adult T-cell leukemia (ATL) with a radiotherapy-resistant bulky disease treated with concomitant radiotherapy and two topoisomerase inhibitors: etoposide (VP-16) and irinotecan (CPT-11). Patient 1 was a 78-year-old man with chemotherapy-resistant inguinal bulky mass. Radiotherapy (total 40 Gy) for this inguinal lesion was started; however, the bulky disease was found to be resistant to radiotherapy and progressed. VP-16 and CPT-11 were administered in addition to radiotherapy (after a total of 20 Gy of radiotherapy). Patient 2 was a 71-year-old man with a solitary bulky mass in left cervical lesion. Various previous chemotherapy and radiotherapy approaches had not been able to control the disease. Six months after first radiotherapy, the bulky disease rapidly progressed with the occurrence of pain. Second radiotherapy (30 Gy) was started with simultaneous administration of CPT-11 and VP-16. In both cases, the bulky disease gradually regressed and completely disappeared by the end of radiotherapy. Thus, flexible adaptation of concomitant chemoradiotherapy including two topoisomerase inhibitors may offer a potential therapeutic option for radiotherapy-resistant bulky diseases, even in hematological malignancies.

  12. Structure of adduct X, the last unknown of the six major DNA adducts of mitomycin C formed in EMT6 mouse mammary tumor cells.

    PubMed

    Palom, Y; Belcourt, M F; Musser, S M; Sartorelli, A C; Rockwell, S; Tomasz, M

    2000-06-01

    Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC-DNA adducts. We identified the last unknown of these ("adduct X") as a guanine N(2) adduct of 2, 7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N(2). The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-(3)H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 microg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM-guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC-DNA adducts, the 2,7-DAM-DNA adducts are not cytotoxic.

  13. Biological activities of eco-friendly synthesized Hantzsch adducts.

    PubMed

    Pacheco, Samira R; Braga, Taniris C; da Silva, Daniel L; Horta, Livia P; Reis, Fabiano S; Ruiz, Ana Lucia T G; de Carvalho, Joao E; Modolo, Luzia V; de Fatima, Ângelo

    2013-09-01

    Fourteen Hantzsch adducts with different substituents at the C-4 position were synthesized through multicomponent reactions by using citric or lactic acid as catalysts. To the best of our knowledge, this is the first report on the synthesis of such a class of compounds based on multicomponent reactions catalyzed by non-toxic organic acids. The potential to scavenge reactive nitrogen/oxygen species (RNS/ROS) and the ability to inhibit cancer cell growth were then investigated. Among the synthesized compounds, adduct 15 was the most promising free radical scavenger, while adduct 20 was shown to have a wider spectrum of action on the cancer cells studied. These results highlight Hantzsch adducts as lead compounds for obtaining new free radical scavengers and anticancer agents.

  14. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    SciTech Connect

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T.; Pan, Chong-xian

    2015-11-06

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). In conclusion, this rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

  15. Mass spectrometric analysis of tobacco-specific hemoglobin adducts.

    PubMed Central

    Schäffler, G; Betz, C; Richter, E

    1993-01-01

    Hemoglobin adducts of the common metabolite of the tobacco-specific nitrosamine (TSNA) 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and of 4-aminobiphenyl (4-ABP) were quantified in blood samples from smokers and nonsmokers to test their suitability for biomonitoring tobacco smoke exposure. Additionally, TSNA adducts were measured in nasal snuff users. Mild alkaline treatment of hemoglobin releases 4-ABP and HPB, which were analyzed in parallel by capillary gas chromatography with electronic impact or negative ion chemical-ionization mass spectrometry (EI- or NICI-GC-MS). Samples of snuff users showed high levels of HPB adducts not correlated with the amount or type of snuff used. HPB concentrations in smokers and nonsmokers, however, were much lower, with no group-specific differences detectable. In contrast, 4-ABP adduct levels were much higher in smokers than in nonsmokers, confirming the significant difference between these two groups reported by others. PMID:8319620

  16. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    PubMed Central

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T.; Pan, Chong-xian

    2016-01-01

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic. PMID:26544157

  17. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    DOE PAGES

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; ...

    2015-11-06

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). In conclusion, this rapid report provides themore » first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.« less

  18. Coordination complexes of niobium and tantalum pentahalides with a bulky NHC ligand.

    PubMed

    Bortoluzzi, Marco; Ferretti, Eleonora; Marchetti, Fabio; Pampaloni, Guido; Zacchini, Stefano

    2016-04-28

    The 1 : 1 molar reactions of niobium and tantalum pentahalides with the monodentate NHC ligand 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene (Ipr), in toluene (or benzene) at ca. 80 °C, afforded the complexes NbX5(Ipr) (X = F, ; Br, ) and TaX5(Ipr) (X = F, ; Cl, ; Br, ), in generally good yields. Complexes represent uncommon cases of stable NHC adducts of metal halides with the metal in an oxidation state higher than +4, and also rare examples of Nb-NHC and Ta-NHC bonding systems. In particular, the X-ray molecular structure determined for provides the unprecedented crystallographic characterization of a tantalum compound with a monodentate NHC ligand. DFT results indicate that the metal-carbon bond in is a purely σ one. According to NMR studies ((1)H, (13)C, (93)Nb), the formation of , , , as well as the previously communicated NbCl5(Ipr), , proceeded with the intermediacy of [MX6](-) salts, presumably due to steric reasons. On the other hand, the intermediate formation of MF6(-) in the pathways to and was not observed, according to (19)F (and (93)Nb in the case of ) NMR. DFT calculations were carried out in order to shed light on structural and mechanistic aspects, and allowed to trace possible reaction routes.

  19. Condensed tannin-resorcinol adducts in laminating adhesives

    Treesearch

    Richard W. Hemingway; Roland E. Kreibich

    1985-01-01

    A condensed tannin-resorcinol adduct made by co-reaction of an extract from southern pine bark with resorcinol at a 2 to 1 weight ratio was used to prepare a laminating resin in which the entire amount of resorcinol normally used was replaced by this adduct. The resin was formulated into a room temperature setting adhesive that meets the basic criteria of product...

  20. Arytenoid adduction combined with Gore-Tex medialization thyroplasty.

    PubMed

    McCulloch, T M; Hoffman, H T; Andrews, B T; Karnell, M P

    2000-08-01

    To describe the technique of combined Gore-Tex medialization thyroplasty with arytenoid adduction and to determine the long-term vocal outcome of patients treated for unilateral vocal cord paralysis with this procedure. A retrospective chart review and patient reevaluation for patients treated at The University of Iowa Hospitals and Clinics between May 1995 and June 1999. The review addressed patient demographics, perioperative and long-term complications, and voice outcomes. Details of the surgical technique are provided within the manuscript. Seventy-two Gore-Tex medialization procedures were completed. Arytenoid adduction was included in 22 of these procedures. This subset of patients was compared with the patients treated with Gore-Tex alone. No major postoperative complications occurred in either group. Preoperative and postoperative voice and videostroboscopy data were available for 19 arytenoid adduction patients and 25 Gore-Tex alone patients. On a seven-point scale (6 [severely abnormal] --> 0 [normal voice]), the average patient rating of voice dysfunction improved from 4.2 to 1.6 (arytenoid adduction) and 4.5 to 2.8 (Gore-Tex alone). Maximum phonation time improved from 6.9 seconds to 16.7 seconds in the arytenoid adduction group. Subjective voice assessment employing the four-point GRBAS scale (3 [severely abnormal] --> 0 [normal]) identified average improvement from an overall grade of 2.1 to 0.8 arytenoid adduction and 2.2 to 1.5 in the Gore-Tex alone group. Improvement was identified in the vocal quality of breathiness from 1.9 to 0.2 (arytenoid adduction) and 1.9 to 0.9 (Gore-Tex alone). The combined technique of Gore-Tex medialization thyroplasty and arytenoid adduction provide functional results that appear to exceed the improvement attained with medialization alone.

  1. ACCUMULATION OF M1DG DNA ADDUCTS AFTER ...

    EPA Pesticide Factsheets

    ABSTRACT: Oxidative DNA damage is one of the key events leading to mutation and cancer. The present study examined the accumulation of M1dG DNA adducts, 3-(2’-deoxy-β-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, after single or yearly exposure to polyhalogenated aromatic hydrocarbons (PHAH) in order to test the role of oxidative DNA damage in PHAH carcinogenicity. The effect of PHAH exposure on the number of M1dG adducts was explored initially in female mice exposed to a single dose of either TCDD or a PHAH mixture. This study demonstrated that a single exposure to PHAH had no significant effect on the number of M1dG adducts compared to the corn oil control group. The role of M1dG adducts in PCB-induced carcinogenicity was further investigated in rats exposed for a year to PCB 153, PCB 126, or a mixture of the two. PCB 153 had no significant effect on M1dG adducts number in liver and brain tissues from the exposed rats compared to controls. However, high dose PCB 126 exposure resulted in M1dG adducts accumulation in the liver. More importantly, starting at low doses, co-administration of equal proportions of PCB 153 and PCB 126 resulted in dose-dependent increases in M1dG adducts accumulation in the liver. Interestingly, the result from co-administration of different amounts of PCB 153 with fixed amounts of PCB 126 demonstrated more M1dG adducts accumulation with higher doses of PCB 153. These results are consistent with the results from canc

  2. [Hemoglobin adducts as biomarkers of human exposure to selected xenobiotics].

    PubMed

    Bukowska, Bożena

    2015-06-12

    In the living and working environments more and more new substances of anthropogenic origin exerting toxic properties appear. Simultaneously, the evaluation of human exposure is assessed. For many years adducts of hemoglobin (Hb) have been useful markers of the exposure of humans to various xenobiotics. These adducts are also termed biologically effective dose biomarkers. This paper focuses on a review of literature, mainly from the years 2010-2014, which refers to the hemoglobin adducts of toxic compounds with electrophilic properties. In the interactions of xenobiotics with hemoglobin, groups such as thiol, amino, carboxyl and hydroxyl of this hemoprotein are involved. These combinations occur most often in the reaction of xenobiotics with an N-terminal amino group of valine in Hb, imidazole nitrogen of histidine and cysteine sulfhydryl β93. Hb adducts are characterized by high availability, a long period of occurrence (up to 120 days) in the circulatory system, and high durability, and they have contact with all cells of the body. The measurement of hemoglobin adducts can be potentially used in the assessment of exposure to many xenobiotics such as acrylamide; substances present in tobacco smoke, e.g. benzo(α)pyrene and benzanthracene, ethylene oxide, aryl amines; and substances used on a large scale in industry such as glycidol and naphthalene and its derivatives. Recently the possibility of determination of hemoglobin adducts with estrogen metabolites has been postulated as indicators informing about heightened risk of breast cancer. Protein adducts are used as an alternative to DNA adducts for different classes of electrophilic substances.

  3. Structural and Dynamic Characterization of Polymerase κ’s Minor Groove Lesion Processing Reveals How Adduct Topology Impacts Fidelity

    PubMed Central

    2015-01-01

    DNA lesion bypass polymerases process different lesions with varying fidelities, but the structural, dynamic, and mechanistic origins of this phenomenon remain poorly understood. Human DNA polymerase κ (Polκ), a member of the Y family of lesion bypass polymerases, is specialized to bypass bulky DNA minor groove lesions in a predominantly error-free manner, by housing them in its unique gap. We have investigated the role of the unique Polκ gap and N-clasp structural features in the fidelity of minor groove lesion processing with extensive molecular modeling and molecular dynamics simulations to pinpoint their functioning in lesion bypass. Here we consider the N2-dG covalent adduct derived from the carcinogenic aromatic amine, 2-acetylaminofluorene (dG-N2-AAF), that is produced via the combustion of kerosene and diesel fuel. Our simulations reveal how the spacious gap directionally accommodates the lesion aromatic ring system as it transits through the stages of incorporation of the predominant correct partner dCTP opposite the damaged guanine, with preservation of local active site organization for nucleotidyl transfer. Furthermore, flexibility in Polκ’s N-clasp facilitates the significant misincorporation of dTTP opposite dG-N2-AAF via wobble pairing. Notably, we show that N-clasp flexibility depends on lesion topology, being markedly reduced in the case of the benzo[a]pyrene-derived major adduct to N2-dG, whose bypass by Polκ is nearly error-free. Thus, our studies reveal how Polκ’s unique structural and dynamic properties can regulate its bypass fidelity of polycyclic aromatic lesions and how the fidelity is impacted by lesion structures. PMID:25148552

  4. Adduction of untested derived stimulus relations depends on environmental complexity.

    PubMed

    Rippy, Sterling M; Doughty, Adam H

    2017-10-01

    The present research assessed adduction involving derived stimulus relations as a function of environmental complexity. In Group CA, four college students were trained with arbitrary-matching-to-sample discriminations that could have established four, 3-member stimulus classes. In Group EA, four other students were trained with discriminations that could have established four, 5-member classes. Neither group received derived-relations testing; instead, adduction was assessed immediately after the baseline discriminations were learned. The adduction assessment required participants to derive the untested CA (Group CA) or EA (Group EA) equivalence relations and combine them with their already learned math skills. All participants in Group CA showed above 90% accuracy during the adduction assessment, whereas only one of four Group EA participants responded in that manner. These results extend adduction to untested equivalence relations and clarify the environmental conditions under which such adduction is less likely to occur (i.e., with larger relational networks). Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Detection of adriamycin-DNA adducts by accelerator mass spectrometry.

    PubMed

    Coldwell, Kate; Cutts, Suzanne M; Ognibene, Ted J; Henderson, Paul T; Phillips, Don R

    2010-01-01

    There have been many attempts in the past to determine whether significant levels of Adriamycin-DNA adducts form in cells and contribute to the anticancer activity of this agent. Supraclincal drug levels have been required to study drug-DNA adducts because of the lack of sensitivity associated with many of the techniques employed, including liquid scintillation counting of radiolabeled drug. The use of accelerator mass spectrometry (AMS) has provided the first direct evidence of Adriamycin-DNA adduct formation in cells at clinically relevant Adriamycin concentrations. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection (compared to liquid scintillation counting) and has revealed adduct formation within an hour of drug treatment. The rigorous protocol required for this approach, together with many notes on the precautions and procedures required in order to ensure that absolute levels of Adriamycin-DNA adducts can be determined with good reproducibility, is outlined in this chapter.

  6. Insight on mendable resin made by combining Diels-Alder epoxy adducts with DGEBA

    NASA Astrophysics Data System (ADS)

    Dello Iacono, S.; Martone, A.; Filippone, G.; Acierno, D.; Zarrelli, M.; Giordano, M.; Amendola, E.

    2016-05-01

    Formation of micro-cracks is a critical problem in polymers and polymer composites during their service in structural applications. In this context, materials endowed with self-healing features would lead to the next polymers generation. In the present paper, an epoxy system integrating Diels-Alder epoxy adducts is investigated by thermal and spectroscopic analysis. The direct and retro D-A reaction have been studied by FTIR and specific absorption bands have been identified. Finally, mechanical tests have been performed on the system. The polymer is able to heal fracture and micro-cracks recovering its stiffness after a thermal treatment.

  7. A treatment planning approach to spatially fractionated megavoltage grid therapy for bulky lung cancer

    SciTech Connect

    Costlow, Heather N.; Zhang, Hualin; Das, Indra J.

    2014-10-01

    The purpose of this study was to explore the treatment planning methods of spatially fractionated megavoltage grid therapy for treating bulky lung tumors using multileaf collimator (MLC). A total of 5 patients with lung cancer who had gross tumor volumes ranging from 277 to 635 cm{sup 3} were retrospectively chosen for this study. The tumors were from 6.5 to 9.6 cm at shortest dimension. Several techniques using either electronic compensation or intensity-modulated radiation therapy (IMRT) were used to create a variety of grid therapy plans on the Eclipse treatment planning system. The dose prescription point was calculated to the volume, and a dose of 20 Gy with 6-MV/15-MV beams was used in each plan. The dose-volume histogram (DVH) curves were obtained to evaluate dosimetric characteristics. In addition, DVH curves from a commercially available cerrobend grid collimator were also used for comparison. The linear-quadratic radiobiological response model was used to assess therapeutic ratios (TRs) and equivalent uniform doses (EUD) for all generated plans. A total of 6 different grid therapy plans were created for each patient. Overall, 4 plans had different electronic compensation techniques: Ecomps-Tubes, Ecomps-Circles, Ecomps-Squares, and Ecomps-Weave; the other 2 plans used IMRT and IMRT-Weave techniques. The DVH curves and TRs demonstrated that these MLC-based grid therapy plans can achieve dosimetric properties very similar to those of the cerrobend grid collimator. However, the MLC-based plans have larger EUDs than those with the cerrobend grid collimator. In addition, the field shaping can be performed for targets of any shape in MLC-based plans. Thus, they can deliver a more conformal dose to the targets and spare normal structures better than the cerrobend grid collimator can. The plans generated by the MLC technique demonstrated the advantage over the standard cerrobend grid collimator on accommodating targets and sparing normal structures. Overall, 6

  8. A mitomycin-N6-deoxyadenosine adduct isolated from DNA.

    PubMed

    Palom, Y; Lipman, R; Musser, S M; Tomasz, M

    1998-03-01

    A minor N6-deoxyadenosine adduct of mitomycin C (MC) was isolated from synthetic oligonucleotides and calf thymus DNA, representing the first adduct of MC and a DNA base other than guanine. The structure of the adduct (8) was elucidated using submilligram quantities of total available material. UV difference spectroscopy, circular dichroism, and electrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligonucleotides was designed to probe the specificities of the alkylation of adenine by MC. The nature and frequency of the oligonucleotide-MC adducts formed under conditions of reductive activation of MC were determined by their enzymatic digestion to the nucleoside level followed by quantitative analysis of the products by HPLC. The analyses indicated the following: (i) (A)n sequence is favored over (AT)n for adduct formation; (ii) the alkylation favors the duplex structure; (iii) at adenine sites only monofunctional alkylation occurs; (iv) the adenine-to-alkylation frequency in the model oligonucleotides was 0.3-0.6 relative to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 relative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The overall ratio of adenine to guanine alkylation in calf thymus DNA was 0.03, indicating that 8 is a minor MC-DNA adduct relative to MC-DNA adducts at guanine residues in the present experimental residues in the present experimental system. However, the HPLC elution time of 8 coincides with that of a major, unknown MC adduct detected previously in mouse mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chowdary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockwell, S., and Tomasz, M. (1993) Cancer Res. 53, 5127-5134]. Thus, 8 may be identical or closely related to this major adduct formed in vivo. This possibility can now be tested by

  9. Molecular Epidemiology of Breast Cancer: Development and Validation of Acetylation Methods for Carcinogen-DNA Adduct Detection

    DTIC Science & Technology

    2001-10-01

    epidemiological studies, and determine adduct levels in relation to metabolizing gene polymorphisms . The originally proposed assay is novel because one uses a...carcinogenic mechanisms. Currently, many ongoing breast cancer studies are exploring risks related to genetic polymorphisms in these genes. Yet these...the surrogate tissue). Finally, in these subjects, we will perform assays for genetic polymorphisms , to assess the association of "at risk" genetic

  10. Partitioning of knee joint internal forces in gait is dictated by the knee adduction angle and not by the knee adduction moment.

    PubMed

    Adouni, M; Shirazi-Adl, A

    2014-05-07

    Medial knee osteoarthritis is a debilitating disease. Surgical and conservative interventions are performed to manage its progression via reduction of load on the medial compartment or equivalently its surrogate measure, the external adduction moment. However, some studies have questioned a correlation between the medial load and adduction moment. Using a musculoskeletal model of the lower extremity driven by kinematics-kinetics of asymptomatic subjects at gait midstance, we aim here to quantify the relative effects of changes in the knee adduction angle versus changes in the adduction moment on the joint response and medial/lateral load partitioning. The reference adduction rotation of 1.6° is altered by ±1.5° to 3.1° and 0.1° or the knee reference adduction moment of 17Nm is varied by ±50% to 25.5Nm and 8.5Nm. Quadriceps, hamstrings and tibiofemoral contact forces substantially increased as adduction angle dropped and diminished as it increased. The medial/lateral ratio of contact forces slightly altered by changes in the adduction moment but a larger adduction rotation hugely increased this ratio from 8.8 to a 90 while in contrast a smaller adduction rotation yielded a more uniform distribution. If the aim in an intervention is to diminish the medial contact force and medial/lateral load ratio, a drop of 1.5° in adduction angle is much more effective (causing respectively 12% and 80% decreases) than a reduction of 50% in the adduction moment (causing respectively 4% and 13% decreases). Substantial role of changes in adduction angle is due to the associated alterations in joint nonlinear passive resistance. These findings explain the poor correlation between knee adduction moment and tibiofemoral compartment loading during gait suggesting that the internal load partitioning is dictated by the joint adduction angle. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence

    PubMed Central

    Jain, Vipin; Hilton, Benjamin; Patnaik, Satyakam; Zou, Yue; Chiarelli, M. Paul; Cho, Bongsup P.

    2012-01-01

    Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and -G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1 > G2). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G2∼G1 > G3, a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts. PMID:22241773

  12. Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE-dual-LIF.

    PubMed

    Zabel, Robert; Kullmann, Maximilian; Kalayda, Ganna V; Jaehde, Ulrich; Weber, Günther

    2015-02-01

    Pt-based anticancer drugs, such as cisplatin, are known to undergo several (bio-)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA-Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA-Pt by using CE-LIF. CE-MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA-Pt are detectable in sensitive and cisplatin-resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA-Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE-LIF detection within 5 min.

  13. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  14. Characterization of deoxyguanosine adducts from hydroquinone/benzoquinone

    SciTech Connect

    Jowa, J.; Winkel, S.; Witz, G.; Snyder, R.

    1986-03-01

    Occupational exposure to benzene has long been associated with the development of pancytopenia and leukemia. This toxicity has been attributed to the action of benzene metabolites. The authors have chosen to investigate the reaction of hydroquinone (HQ)/benzoquinone(BQ) with deoxyguanosine(dG) and DNA. (/sup 14/C)HQ was incubated with (/sup 3/H)dG in potassium phosphate buffer pH7.2 for 24 hours. Two dual labeled products were found by HPLC and presumed to be adducts. The same result was obtained when BQ was substituted in the reaction for HQ. Both adducts were found in isolated DNA from Clostridium perfringens, Micrococcus lysodeikticus, human placenta and calf thymus reacted with HO under similar conditions. One of the dG adducts was proposed to be (/sup 3/'OH) benzetheno(1,N-2)deoxyguanosine based on NMR and mass spectral results. The other adduct was characterized by a molecular weight of 339. The latter adduct was found in greater amounts than the former when HQ was reacted with denatured DNA.

  15. Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases

    PubMed Central

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-01-01

    Formaldehyde is universally employed to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, and avoiding high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5–2.4 fold using a catalyst under optimized conditions, and by 7–25 fold compared to a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens. PMID:26291948

  16. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases.

    PubMed

    Karmakar, Saswata; Harcourt, Emily M; Hewings, David S; Scherer, Florian; Lovejoy, Alexander F; Kurtz, David M; Ehrenschwender, Thomas; Barandun, Luzi J; Roost, Caroline; Alizadeh, Ash A; Kool, Eric T

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  17. Glutathione Adduct Patterns of Michael-Acceptor Carbonyls.

    PubMed

    Slawik, Christian; Rickmeyer, Christiane; Brehm, Martin; Böhme, Alexander; Schüürmann, Gerrit

    2017-02-22

    Glutathione (GSH) has so far been considered to facilitate detoxification of soft organic electrophiles through covalent binding at its cysteine (Cys) thiol group, followed by stepwise catalyzed degradation and eventual elimination along the mercapturic acid pathway. Here we show that in contrast to expectation from HSAB theory, Michael-acceptor ketones, aldehydes and esters may form also single, double and triple adducts with GSH involving β-carbon attack at the much harder N-terminus of the γ-glutamyl (Glu) unit of GSH. In particular, formation of the GSH-N single adduct contradicts the traditional view that S alkylation always forms the initial reaction of GSH with Michael-acceptor carbonyls. To this end, chemoassay analyses of the adduct formation of GSH with nine α,β-unsaturated carbonyls employing high performance liquid chromatography and tandem mass spectrometry have been performed. Besides enriching the GSH adductome and potential biomarker applications, electrophilic N-terminus functio-nalization is likely to impair GSH homeostasis substantially through blocking the γ-glutamyl transferase catalysis of the first breakdown step of modified GSH, and thus its timely reconstitution. The discussion includes a comparison with cyclic adducts of GSH and furan metabolites as reported in literature, and quantum chemically calculated thermodynamics of hard-hard, hard-soft and soft-soft adducts.

  18. Effect of Michael adduction on peptide preservation in natural waters.

    PubMed

    McKee, G A; Kobiela, M E; Hatcher, P G

    2014-09-20

    The reaction of peptides with chemicals already present in natural waters (such as polycyclic aromatic hydrocarbons) is one method that has been suggested to preserve peptides for the longer term. In this study we test whether the reaction of tetrapeptides with a model quinone can help stabilise the peptide in one polluted riverine system, Elizabeth River in Virginia, USA. We found that there is almost no difference in rate constants between the peptide and its quinone adduct (e.g. 6.62 versus 6.86 per day for AVFA and its respective adduct). However, when monitoring the removal of the adduct from natural water, we identified two new compounds that are formed as a result of its decomposition. Using tandem mass spectrometry we identified potential structures and mechanisms for the formation of these new compounds. These new compounds are more recalcitrant than their parent peptide-quinone adduct, since they remain in solution for 3-10 times longer. Based on our findings we postulate that the reaction of peptides with quinones can help preserve sections of the original peptide following an initial rearrangement of the original adduct, potentially explaining why seemingly labile peptides are observed in most natural waters.

  19. Antitumor Trans Platinum Adducts of GMP and AMP

    PubMed Central

    Liu, Yangzhong; Sivo, Maria F.; Natile, Giovanni

    2000-01-01

    Recently it has been shown that several analogues of the clinically ineffective trans-DDP exhibit antitumor activity comparable to that of cis-DDP. The present paper describes the binding of antitumor trans-[PtCl2(E-iminoether)2] (trans-EE) to guanosinemonophosphate (GMP) and adenosinemonophosphate (AMP). We have used HPLC and 1H and 15N NMR to characterize the different adducts. In the case of a 1:1 mixture of trans-EE and GMP, at an early stage of the reaction, a monofunctional adduct is formed which, subsequently, is partly converted into a monosolvated monofunctional species. After about 70 hours an equilibrium is established between chloro and solvato monofunctional adducts at a ratio of 30/70. In the presence of excess GMP (4:1) the initially formed monofunctional adducts react further to give two bifunctional adducts, one with the iminoether ligands in their original E configurations and the other with the iminoether ligands having one E and the other, Z configurations. The coordination geometry obtained by energy minimization calculations is in qualitative agreement with 2D NMR data. PMID:18475942

  20. PROTEIN ADDUCTS AS BIOMAKERS OF EXPOSURE TO ORGANOPHOSPHORUS COMPOUNDS

    PubMed Central

    Marsillach, Judit; Costa, Lucio G.; Furlong, Clement E.

    2013-01-01

    Exposure to organophosphorus (OP) compounds can lead to serious neurological damage or death. Following bioactivation by the liver cytochromes P450, the OP metabolites produced are potent inhibitors of serine active-site enzymes including esterases, proteases and lipases. OPs may form adducts on other cellular proteins. Blood cholinesterases (ChEs) have long served as biomarkers of OP exposure in humans. However, the enzymatic assays used for biomonitoring OP exposures have several drawbacks. A more useful approach will focus on multiple biomarkers and avoid problems with the enzymatic activity assays. OP inhibitory effects result from a covalent bond with the active-site serine of the target enzymes. The serine OP adducts become irreversible following a process referred to as aging where one alkyl group dissociates over variable lengths of time depending on the OP adduct. The OP-adducted enzyme then remains in circulation until it is degraded, allowing for a longer window of detection compared with direct analysis of OPs or their metabolites. Mass spectrometry (MS) provides a very sensitive method for identification of post-translational protein modifications. MS analyses of the percentage adduction of the active-site serine of biomarker proteins such as ChEs will eliminate the need for basal activity levels of the individual and will provide for a more accurate determination of OP exposure. MS analysis of biomarker proteins also provides information about the OP that has caused inhibition. Other useful biomarker proteins include other serine hydrolases, albumin, tubulin and transferrin. PMID:23261756

  1. Perinephric stranding and bulky psoas mimicking pyelonephritis in a case of non-Hodgkin lymphoma of kidney.

    PubMed

    Singh, Shrawan Kumar; Sharma, Aditya Prakash; Mittal, Ankur; Lal, Anupam

    2015-05-01

    A 68-year-old male patient presented with fever and right groin pain. He had leukocytosis with azotemia. Computed tomography revealed enlarged right kidney with thickening and enhancement of walls of pelvicalyceal system and perinephric fat stranding, suggestive of pyelonephritis. Multiple enlarged lymph nodes encased right renal vessels and were present in the retrocaval region. The right psoas muscle was bulky. Fine-needle aspiration cytology and biopsy from the lesions showed features of non-Hodgkin lymphoma. Immunohistochemistry confirmed the diagnosis of diffuse, large, B-cell lymphoma. We emphasize lymphoma in differential diagnosis of atypical renal imaging suggestive of pyelonephritis and perinephritis.

  2. Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

    PubMed

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2012-12-04

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

  3. Conformational preferences of DNA following damage by aristolochic acids: Structural and energetic insights into the different mutagenic potential of the ALI and ALII-N(6)-dA adducts.

    PubMed

    Kathuria, Preetleen; Sharma, Purshotam; Abendong, Minette N; Wetmore, Stacey D

    2015-04-21

    Aristolochic acids (AAI and AAII), produced by the Aristolochiaceae family of plants, are classified as group I (human) carcinogens by the International Agency for Research on Cancer. These acids are metabolized in cells to yield aristolactams (ALI and ALII, respectively), which further form bulky adducts with the purine nucleobases. Specifically, the adenine lesions are more persistent in cells and have been associated with chronic renal diseases and related carcinogenesis. To understand the structural basis of the nephrotoxicity induced by AAs, the ALI-N(6)-dA and ALII-N(6)-dA lesions are systematically studied using computational methods. Density functional theory calculations indicate that the aristolactam moiety intrinsically prefers a planar conformation with respect to adenine. Nucleoside and nucleotide models suggest that the anti and syn orientations about the glycosidic bond are isoenergetic for both adducts. Molecular dynamics simulations and free energy calculations reveal that the anti base-displaced intercalated conformation is the most stable conformer for both types of AL-N(6)-dA adducted DNA, which agrees with previous experimental work on the ALII-N(6)-dA adduct and thereby validates our approach. Interestingly, this conformer differs from the dominant conformations adopted by other N6-linked adenine lesions, including those derived from polycyclic aromatic hydrocarbons. Furthermore, the second most stable syn base-displaced intercalated conformation lies closer in energy to the anti base-displaced intercalated conformation for ALI-N(6)-dA compared to ALII-N(6)-dA. This indicates that a mixture of conformations may be detectable for ALI-N(6)-dA in DNA. If this enhanced conformational flexibility of double-stranded DNA persists when bound to a lesion-bypass polymerase, this provides a possible structural explanation for the previously observed greater nephrotoxic potential for the ALI versus ALII-N(6)-dA adduct. In addition, the structural

  4. NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

    PubMed Central

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2012-01-01

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

  5. Site-Specific Protein Adducts of 4-Hydroxy-2(E)-Nonenal in Human THP-1 Monocytic Cells: Protein Carbonylation Is Diminished by Ascorbic Acid

    SciTech Connect

    Chavez, Juan; Chung, Woon-Gye; Miranda, Cristobal L.; Singhal, Mudita; Stevens, Jan F.; Maier, Claudia S.

    2010-01-18

    The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multipronged proteomic approach involving electrophoretic, immunoblotting, and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction, and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses, and enzymes of the glycolytic and other metabolic pathways. Finally, this study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin α-1B chain, Cys-351 and Cys-499 in α-actinin-4, Cys-328 in vimentin, Cys-369 in d-3-phosphoglycerate dehydrogenase, and His-246 in aldolase A.

  6. DNA adducts: mass spectrometry methods and future prospects.

    PubMed

    Farmer, P B; Brown, K; Tompkins, E; Emms, V L; Jones, D J L; Singh, R; Phillips, D H

    2005-09-01

    Detection of DNA adducts is widely used for the monitoring of exposure to genotoxic carcinogens. Knowledge of the nature and amounts of DNA adducts formed in vivo also gives valuable information regarding the mutational effects that may result from particular exposures. The power of mass spectrometry (MS) to achieve qualitative and quantitative analyses of human DNA adducts has increased greatly in recent years with the development of improved chromatographic interfaces and ionisation sources. Adducts have been detected on nucleic acid bases, 2'-deoxynucleosides or 2'-deoxynucleotides, with LC-MS/MS being the favoured technique for many of these analyses. Our current applications of this technique include the determination of N7-(2-carbamoyl-2-hydroxyethyl)-guanine, which was postulated to be found as a DNA repair product in urine following exposure to acrylamide, and of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyadenosine, as markers of oxidative damage in human lymphocyte DNA. Higher sensitivity (with a detection limit of 1-10 adducts/10(12) nucleotides) may be achieved by the use of accelerator mass spectrometry (AMS), although this requires the presence of certain isotopes, such as [(14)C], in the material being analysed. In order to make this technique more amenable for studies of human exposure to environmental carcinogens, new postlabelling techniques, incorporating [(14)C] into specific DNA adducts after formation, are being developed. It is expected that combining the use of advanced MS techniques with existing (32)P-postlabelling and immunochemical methodologies will contribute greatly to the understanding of the burden of human exposure to environmental carcinogens.

  7. DNA adducts: Mass spectrometry methods and future prospects

    SciTech Connect

    Farmer, P.B. . E-mail: pbf1@le.ac.uk; Brown, K.; Tompkins, E.; Emms, V.L.; Jones, D.J.L.; Singh, R.; Phillips, D.H.

    2005-09-01

    Detection of DNA adducts is widely used for the monitoring of exposure to genotoxic carcinogens. Knowledge of the nature and amounts of DNA adducts formed in vivo also gives valuable information regarding the mutational effects that may result from particular exposures. The power of mass spectrometry (MS) to achieve qualitative and quantitative analyses of human DNA adducts has increased greatly in recent years with the development of improved chromatographic interfaces and ionisation sources. Adducts have been detected on nucleic acid bases, 2'-deoxynucleosides or 2'-deoxynucleotides, with LC-MS/MS being the favoured technique for many of these analyses. Our current applications of this technique include the determination of N7-(2-carbamoyl-2-hydroxyethyl)-guanine, which was postulated to be found as a DNA repair product in urine following exposure to acrylamide, and of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyadenosine, as markers of oxidative damage in human lymphocyte DNA. Higher sensitivity (with a detection limit of 1-10 adducts/10{sup 12} nucleotides) may be achieved by the use of accelerator mass spectrometry (AMS), although this requires the presence of certain isotopes, such as [{sup 14}C], in the material being analysed. In order to make this technique more amenable for studies of human exposure to environmental carcinogens, new postlabelling techniques, incorporating [{sup 14}C] into specific DNA adducts after formation, are being developed. It is expected that combining the use of advanced MS techniques with existing {sup 32}P-postlabelling and immunochemical methodologies will contribute greatly to the understanding of the burden of human exposure to environmental carcinogens.

  8. Case report: impressive response to pembrolizumab in a patient with mismatch-repair deficient metastasized colorectal cancer and bulky disease

    PubMed Central

    Kieler, Markus; Scheithauer, Werner; Zielinski, Christoph C; Chott, Andreas; Al-Mukhtar, Ali; Prager, Gerald W

    2016-01-01

    Here, we report the history of a 42-year-old female patient with sporadic mismatch-repair-deficient metastatic colorectal cancer and abdominal bulky disease, who received pembrolizumab (200 mg every 3 weeks) after the failure of third-line treatment. Restaging 3 months after initiation of treatment revealed a striking response with shrinkage of the bulky peritoneal tumour mass (baseline size 11×11×14 cm) to nearly 25% of the original tumour volume (6.2×7.1×10.4 cm). Restaging 8 months after initiation showed further downsizing of the tumour mass (5.5×7.0×8.0 cm). Tumour markers CEA and CA 19-9 decreased to normal levels, haemoglobin level increased from 8 to 13 mg/dL and her overall clinical performance status increased from ECOG 3 to 1 within 3 months. Therapy with pembrolizumab was continued and is still ongoing. We emphasise the importance of testing for mismatch-repair status in metastatic disease. PMID:28255450

  9. Supramolecular chirality induction in bis(zinc porphyrin) by amino acid derivatives: rationalization and applications of the ligand bulkiness effect.

    PubMed

    Borovkov, V V; Yamamoto, N; Lintuluoto, J M; Tanaka, T; Inoue, Y

    2001-06-01

    The achiral syn conformer (face-to-face) of the ethane-bridged bis(zinc porphyrin) (syn-ZnD) transforms into the corresponding chiral extended anti bis-ligated species (anti-ZnD.L2) in the presence of enantiopure ligands (L: amino acid derivatives). The mechanism of the supramolecular chirality induction is based on chiral ligand binding to zinc porphyrins and subsequent formation of either right- or left-handed screw structures in anti-ZnD.L2. The screw structure formation arises from steric interactions between the bulkiest substituent at the asymmetric carbon of the ligand and the peripheral ethyl groups of the neighboring porphyrin ring directed towards the covalent bridge. The sign and amplitude of the induced circular dichroism (CD) are dependent on the steric bulk of the substituents at the chiral center. The greater difference in size between the chiral center's substituents gives the stronger induced CD signal. Rationalization of the ligand bulkiness effect on chirality induction by amino acid derivatives, application of this supramolecular system for the determination of ligand absolute configuration, and relative bulkiness of the substituents at the asymmetric carbon are discussed. Copyright 2001 Wiley-Liss, Inc.

  10. Malondialdehyde-acetaldehyde-adducted protein inhalation causes lung injury.

    PubMed

    Wyatt, Todd A; Kharbanda, Kusum K; McCaskill, Michael L; Tuma, Dean J; Yanov, Daniel; DeVasure, Jane; Sisson, Joseph H

    2012-02-01

    In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde-acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 (IL-8) via the activation of protein kinase C epsilon (PKCɛ). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30μL of 50μg/mL bovine serum albumin (BSA)-MAA, or unadducted BSA for up to 3 weeks. Likewise, human lung surfactant proteins A and D (SPA and SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA-MAA, SPD-MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCɛ activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in unadducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA-MAA or SPD-MAA for 3 weeks, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA-MAA and SPD-MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, keratinocyte chemokine, which is a functional homologue to human IL-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCɛ. These data support that the MAA-adducted protein induces a proinflammatory response in the lungs and

  11. Quantitation of DNA Adducts Induced by 1,3-Butadiene

    NASA Astrophysics Data System (ADS)

    Sangaraju, Dewakar; Villalta, Peter W.; Wickramaratne, Susith; Swenberg, James; Tretyakova, Natalia

    2014-07-01

    Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI+-HRMS3 analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 106 nucleotides in HT1080 cells treated with 0.5-10 μM DEB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 106 nucleotides, respectively. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI+-HRMS3 Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

  12. Chemistry and Biology of Aflatoxin-DNA Adducts

    SciTech Connect

    Stone, Michael P.; Banerjee, Surajit; Brown, Kyle L.; Egli, Martin

    2012-03-27

    Aspergillus flavus is a fungal contaminant of stored rice, wheat, corn, and other grainstuffs, and peanuts. This is of concern to human health because it produces the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}), which is genotoxic and is implicated in the etiology of liver cancer. AFB{sub 1} is oxidized in vivo by cytochrome P450 to form aflatoxin B{sub 1} epoxide, which forms an N7-dG adduct (AFB{sub 1}-N7-dG) in DNA. The latter rearranges to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative that equilibrates between {alpha} and {beta} anomers of the deoxyribose. In DNA, both the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts intercalate above the 5'-face of the damaged guanine. Each produces G {yields} T transversions in Escherichia coli, but the AFB{sub 1}-{beta}-FAPY adduct is more mutagenic. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) provides a model for understanding error-prone bypass of the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts. It bypasses the AFB{sub 1}-N7-dG adduct, but it conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including mis-insertion of dATP, consistent with the G {yields} T mutations characteristic of AFB{sub 1} mutagenesis in E. coli. Crystallographic analyses of a series of binary and ternary complexes with the Dpo4 polymerase revealed differing orientations of the N7-C8 bond of the AFB{sub 1}-N7-dG adduct as compared to the N{sup 5}-C8 bond in the AFB{sub 1}-{beta}-FAPY adduct, and differential accommodation of the intercalated AFB{sub 1} moieties within the active site. These may modulate AFB{sub 1} lesion bypass by this polymerase.

  13. A Cyclic Disilylated Stannylene: Synthesis, Dimerization, and Adduct Formation

    PubMed Central

    2011-01-01

    Reaction of 1,4-dipotassio-1,1,4,4-tetrakis(trimethylsilyl)tetramethyltetrasilane with [(Me3Si)2N]2Sn led to the formation of an endocyclic distannene via the dimerization of a transient stannylene. In the presence of strong donor molecules such as PEt3, the stannylene could be trapped as adduct. Reaction of the PEt3 derivative with B(C6F5)3 gave rise to the formation of the stannylene B(C6F5)3 adduct. PMID:21438553

  14. Laboratory studies of weakly bound adducts of atmospheric interest

    SciTech Connect

    Wine, P.H.; Nicovich, J.M.; Stickel, R.E.; Hynes, A.J.

    1995-12-31

    It is now well-established that weakly bound adducts, i.e., species whose life-times toward unimolecular decomposition are only fractions of a second under atmospheric conditions, play an important role in tropospheric sulfur chemistry. In this presentation, recent results from our laboratory concerning the existence and atmospheric fates of two such weakly bound species, (CH{sub 3}){sub 2}S-OH and (CH{sub 3}){sub 2}S-Cl, will be discussed. In addition, evidence for the formation of weakly bound adducts in reactions of chlorine atoms with methyl halides will be presented.

  15. Possible rare congenital dysinnervation disorder: congenital ptosis associated with adduction.

    PubMed

    Mendes, Sílvia; Beselga, Diana; Campos, Sónia; Neves, Arminda; Campos, Joana; Carvalho, Sílvia; Silva, Eduardo; Castro Sousa, João Paulo

    2015-01-01

    Ptosis is defined as an abnormally low position of the upper eyelid margin. It can be congenital or acquired, uni or bilateral, and isolated or associated with other ocular and nonocular defects. We report a case of a female child, aged 8 years, with congenital right ptosis increased on right adduction and with left ptosis on left adduction. There was no horizontal ocular movement limitation. Apparent underaction of the right inferior oblique muscle was also present. We believe that within the possible mechanisms it is more likely that it is a congenital innervation dysgenesis syndrome (CID)/congenital cranial dysinnervation disorder (CCDD).

  16. Strategy for identifying unknown hemoglobin adducts using adductome LC-MS/MS data: Identification of adducts corresponding to acrylic acid, glyoxal, methylglyoxal, and 1-octen-3-one.

    PubMed

    Carlsson, Henrik; Törnqvist, Margareta

    2016-06-01

    Electrophilic compounds have the ability to form adducts with nucleophilic sites in proteins and DNA in tissues, and thereby constitute risks for toxic effects. Adductomic approaches are developed for systematic screening of adducts to DNA and blood proteins, with the aim to detect unknown internal exposures to electrophiles. In a previous adductomic screening of adducts to N-terminals in hemoglobin, using LC-MS/MS, 19 unknown adducts were detected in addition to seven previously identified adducts. The present paper describes the identification of four of these unknown adducts, as well as the strategy used to identify them. Using LC-MS data from the screening, hypotheses about adduct identities were formulated: probable precursor electrophiles with matching molecular weights were suggested based on the molecular weights of the modifications and the retention times of the analytes, in combination with comparisons of theoretical Log P calculations and databases. Reference adducts were generated by incubation of blood samples with the hypothesized precursor electrophiles. The four identified precursor electrophiles, corresponding to the observed unknown adducts, were glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. Possible origins/exposure sources and toxicological information concerning the electrophilic precursors are discussed. The identified adducts could be explored as possible biomarkers for exposure.

  17. Protein adduct species in muscle and liver of rats following acute ethanol administration.

    PubMed

    Patel, Vinood B; Worrall, Simon; Emery, Peter W; Preedy, Victor R

    2005-01-01

    Previous immunohistochemical studies have shown that the post-translational formation of aldehyde-protein adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde-malondialdehyde-protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)-protein adducts. Furthermore, acetaldehyde-protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed after acute alcohol exposure and that unreduced adducts predominate. Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER-protein adducts. These were used to assay different adduct species in soleus (type I fibre-predominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A] saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal antibody. Furthermore, there was no detectable increase in malondialdehyde-acetaldehyde and HER-protein adducts. Similar results were obtained in the liver. Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.

  18. UNUSUALLY STABLE ADDUCT BETWEEN METHANOLYZED AMOXICILLIN OR AMPICILLIN AND THEIR DIKETOPIPERAZINE DERIVATIVES.

    PubMed

    Kosińska, Katarzyna; Frański, Rafał; Frańska, Magdalena

    2016-01-01

    Amoxicillin and ampicillin were subjected to methanolysis. As expected, the methanolysis products were observed by HPLC-ESI-MS. Besides these products, diketopiperazine derivatives were also detected. Additionally, unusually stable adduct formed between the products of methanolysis and diketopiperazine derivatives was also identified. Analogical adducts were detected when ethanolysis was performed instead of methanolysis. HPLC-ESI-MS analysis of the separated adducts confirmed that the adducts were composed of methanolysis products and diketopiperazine derivatives.

  19. Immunochemical and Biochemical Analysis of Adducts to DNA and Proteins After Exposure to Sulfur Mustard

    DTIC Science & Technology

    1993-05-13

    cQ.00o~33/o/ /9 IMMUNOCHEMICAL AND BIOCHEMICAL ANALYSIS OF O• ADDUCTS TO DNA AND PROTEINS AFTER EXPOSURE TO SULFUR MUSTARD _ == M G.P. van der Schans...chosen to define exposure to sulfur mustard (HD), based on immunochemical analysis of adducts of HD to DNA and proteins. These adducts are agent...calibration of the immunochemical assays. Analogous to HD-DNA adducts , reaction products with blood proteins may be used to establish HD exposure. Since it

  20. Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high pressure liquid chromatography coupled with triple quadrupole mass spectrometry.

    PubMed

    Wu, Xu; Zhu, Lin; Ma, Jiang; Ye, Yang; Lin, Ge

    2017-10-25

    Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family, and have a variety of biological activities. There is a great need to comprehensively profile these phytochemicals and to quantify them for monitoring their contents in the herbs and the biological samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response of POPs. By monitoring the solely formed sodium adduct ions [M+Na](+), a method for simultaneous quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et Arn., and in the plasma obtained from the rats treated with this herb. The results demonstrated that adduct ion formation could be optimized for the qualitative and quantitative analysis of POPs, and our developed PI/FS-PIS scanning and sole [M+Na](+) ion monitoring significantly improved the analysis of POPs in both herbal and

  1. 40 CFR 721.6205 - Hexamethylenediamine adduct of substituted piperidinyloxy (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Hexamethylenediamine adduct of... Significant New Uses for Specific Chemical Substances § 721.6205 Hexamethylenediamine adduct of substituted... substance identified generically as hexamethylenediamine adduct of substituted piperidinyloxy (PMN P-99-0510...

  2. 40 CFR 721.6205 - Hexamethylenediamine adduct of substituted piperidinyloxy (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Hexamethylenediamine adduct of... Significant New Uses for Specific Chemical Substances § 721.6205 Hexamethylenediamine adduct of substituted... substance identified generically as hexamethylenediamine adduct of substituted piperidinyloxy (PMN P-99-0510...

  3. 40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under...

  4. Volatile Barium Beta-Diketonate Polyether Adducts. Synthesis, Characterization and Metalorganic Chemical Vapor Deposition

    DTIC Science & Technology

    1991-05-31

    Volatile Barium 13- Diketonate Polyether Adducts.... Synthesis , Characterization and Metalorganic Chemical Vapor Deposition by Robin A. Gardiner...has been approved for public release and sale: its distribution is unlimited. Volatile, Barium B- Diketonate Polyether Adducts. Synthesis ...NO. NO. INO. ACCESSION NO. Arlington, VA 22217 II 11. TITLE (include Security Classification) Volatile Barium B- Diketonate Polyether Adducts

  5. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Ethylene oxide adduct of fatty acid... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  6. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Ethylene oxide adduct of fatty acid... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  7. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Ethylene oxide adduct of fatty acid... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  8. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Ethylene oxide adduct of fatty acid... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  9. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Ethylene oxide adduct of fatty acid... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  10. Conformations of DNA adducts with polycyclic aromatic carcinogens

    SciTech Connect

    Broyde, S.; Hingerty, B.

    1984-01-01

    Minimized semi-empirical potential energy calculations for a number of carcinogen adducts with dCpdG have yielded molecular views of the adduct conformations. The base displaced and Z type conformations of acetylaminofluorene (AAF) adducts to guanine C-8 have been detailed. Model building shows that base displacement causes kinking and denaturation in the B helix, while the Z helix is largely unperturbed by modification with AAF, in agreement with experimental findings. The minor AAF adduct linked to quanine N/sup 2/ can reside at a B-Z junction, with the carcinogen buried in a groove in the Z direction, without causing denaturation. The syn guanine in these modified Z forms could be mutagenic, the lesion escaping repair because the helix is undeformed, while the distorted base-displaced conformers are repaired. Aminofluorene (AF) and 4-aminobiphenyl (ABP) linked to guanine N/sup 2/ are currently believed to be critical lesions. They all have a pair of A or B type low energy states, one of which has base-base stacking with carcinogen at the helix exterior, and a second with carcinogen-base stacking. The two states are easily interconvertible. It is possible that the carcinogen may reside primarily at the unperturbed helix exterior where it escapes repair, but that carcinogen-base stacking may occur at a critical time during replication, leading to a mutation. 49 references, 8 figures.

  11. NMR at the Picomole Level of a DNA Adduct

    PubMed Central

    Kautz, Roger; Wang, Poguang; Giese, Roger W.

    2014-01-01

    We investigate the limit of detection for obtaining NMR data of a DNA adduct using modern microscale NMR instrumentation, once the adduct has been isolated at the pmol level. Eighty nanograms (130 pmol) of a DNA adduct standard, N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene 5′-monophosphate (AAF-dGMP), in 1.5 μL of D2O with 10% methanol-d4, in a vial, was completely picked up as a droplet suspended in a fluorocarbon liquid, and loaded efficiently into a microcoil probe. This work demonstrates a practical manual method of droplet microfluidic sample loading, previously demonstrated using automated equipment, which provides a several-fold advantage over conventional flow injection. Eliminating dilution during injection and confining the sample into the observed volume realizes the full theoretical mass sensitivity of a microcoil, comparable to a micro-cryo probe. With 80 ng, an NMR spectrum acquired over 40 hr showed all of the resonances seen in a standard spectrum of AAF-dGMP, with a S/N of at least 10, despite broadening due to previously-noted effects of conformational exchange. Also a 2D TOCSY spectrum (total correlation spectroscopy) was acquired on 1.6 μg in 18 hr. This work helps to define the utility of NMR in combination with other analytical methods for the structural characterization of a small amount of a DNA adduct. PMID:24028148

  12. Infrared spectroscopy of fullerene C60/anthracene adducts

    NASA Astrophysics Data System (ADS)

    García-Hernández, D. A.; Cataldo, F.; Manchado, A.

    2013-09-01

    Recent Spitzer Space Telescope observations of several astrophysical environments such as planetary nebulae, reflection nebulae and R Coronae Borealis stars show the simultaneous presence of mid-infrared features attributed to neutral fullerene molecules (i.e. C60) and polycyclic aromatic hydrocarbons (PAHs). If C60 fullerenes and PAHs coexist in fullerene-rich space environments, then C60 may easily form adducts with a number of different PAH molecules, at least with catacondensed PAHs. Here we present the laboratory infrared spectra (˜2-25 μm) of C60 fullerene and anthracene Diels-Alder mono- and bis-adducts as produced by sonochemical synthesis. We find that C60/anthracene Diels-Alder adducts display spectral features strikingly similar to those from C60 (and C70) fullerenes and other unidentified infrared emission features. Thus, fullerene adducts - if formed under astrophysical conditions and are stable/abundant enough - may contribute to the infrared emission features observed in fullerene-containing circumstellar/interstellar environments.

  13. 40 CFR 721.4590 - Mannich-based adduct.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    .... Requirements as specified in § 721.80(h). (ii) Release to water. Requirements as specified in § 721.90 (a)(4... reporting. (1) The chemical substance generically identified as a Mannich-based adduct (PMN P-93-66) is...

  14. Theoretical investigations on the formation of nitrobenzanthrone-DNA adducts.

    PubMed

    Arlt, Volker M; Phillips, David H; Reynisson, Jóhannes

    2011-09-07

    3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. The thermochemical formation cascades were calculated for six 3-NBA-derived DNA adducts employing its arylnitrenium ion as precursor using density functional theory (DFT). Clear exothermic pathways were found for four adducts, i.e., 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone, 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone. All four have been observed to be formed in cell-free experimental systems. The formation of N-(2'-deoxyadenosin-8-yl)-3-aminobenzanthrone is predicted to be not thermochemically viable explaining its absence in either in vitro or in vivo model systems. However, 2-(2'-deoxyadenosin-8-yl)-3-aminobenzanthrone, can be formed, albeit not as a major product, and is a viable candidate for an unknown adenine adduct observed experimentally. 2-nitrobenzanthrone (2-NBA), an isomer of 3-NBA, was also included in the calculations; it has a higher abundance in ambient air than 3-NBA, but a much lower genotoxic potency. Similar thermochemical profiles were obtained for the calculated 2-NBA-derived DNA adducts. This leads to the conclusion that enzymatic activation as well as the stability of its arylnitrenium ion are important determinants of 2-NBA genotoxicity.

  15. The relationship between brachytherapy dose and outcome in patients with bulky endocervical tumors treated with radiation alone.

    PubMed

    Eifel, P J; Thoms, W W; Smith, T L; Morris, M; Oswald, M J

    1994-01-01

    To evaluate the relationship between brachytherapy dose and outcome in patients treated with external radiotherapy (40 Gy to the whole pelvis) and intracavitary radium therapy for bulky endocervical tumors. Between 1962 and 1985, 98 patients with Stage IB-IIB bulky endocervical carcinomas (> or = 6 cm in diameter) treated with radiotherapy alone received 40 Gy to the whole pelvis followed by 2 or more intracavitary treatments. Twenty-five patients received < 6000 mg-hr of intracavitary treatment and 73 received > or = 6000 mg-hr (an average dose to point A of approximately 49 Gy). Brachytherapy exposures ranged from 4800-7885 mg-hrs. Patients who received < 6000 mg-hr tended to have unfavorable (narrow) vaginal anatomy (p < 0.01) and to be treated in the later years of the study (p < 0.01). The high-dose group included a somewhat greater proportion of patients with positive lymphangiograms or poor responses to initial external beam treatment. Despite having somewhat more favorable tumors, patients who received less than 6000 mg-hr had a higher rate of pelvic disease recurrence at 5 years (33%) than those who received higher doses (16%) (p = 0.03). Actuarial survival rates at 5 years were 44% and 60% for the low- and high-dose groups, respectively (p = 0.14). Among those who received more than 6000 mg-hr, there was no significant relationship between brachytherapy dose and pelvic disease control. Calculated actuarially, the rate of major (> or = grade 3) complications at 5 years was 23% in the low-dose group and 10% in the high-dose group (p = 0.1). The relatively high incidence of pelvic disease recurrence and complications in patients who receive less than 6000 mg-hr reflects the narrow therapeutic window for complication-free pelvic disease control in patients with bulky central disease and unfavorable normal tissue anatomy. The results also demonstrate a high pelvic control rate and acceptable morbidity in patients with favorable anatomy treated with high

  16. External Beam Radiotherapy Followed by {sup 90}Y Ibritumomab Tiuxetan in Relapsed or Refractory Bulky Follicular Lymphoma

    SciTech Connect

    Burdick, Michael J.; Neumann, Donald; Pohlman, Brad; Reddy, Chandana A.; Tendulkar, Rahul D.; Macklis, Roger

    2011-03-15

    Purpose: We combined external beam radiotherapy (EBRT) with yttrium-90 ibritumomab tiuxetan ({sup 90}Y-IT) in an attempt to improve therapeutic response in patients with relapsed or refractory bulky follicular lymphoma (RRBFL). Methods and Materials: Between February 2006 and September 2007, 11 patients with RRBFL were treated with EBRT followed by {sup 90}Y-IT. Bulky disease (BD) was defined as >5 cm. EBRT was delivered to BD as 2,400 cGy in eight fractions using computed tomography (CT)-based planning. BD was contoured as the gross tumor volume. A planning margin of 1 to 2 cm was added depending on anatomical location. After recovery of complete blood counts (CBC), {sup 90}Y-IT was administered at a dose of 0.3 or 0.4 mCi/kg depending on platelet counts. Hematologic toxicity was monitored through weekly CBC. Response was measured by positron emission tomography/CT or CT 3-4 months after {sup 90}Y-IT. Results: Only 2 patients required prolonged breaks between EBRT and {sup 90}Y-IT. The median time after {sup 90}Y-IT for platelets to recover to >100,000/ml was 55 days (range, 41-128 days). Platelet counts for 1 patient, who had received 4 previous chemotherapy regimens, never reached 100,000/ml. The complete and overall responses to combined therapy as measured 3-4 months after {sup 90}Y-IT were 64%. No patients relapsed within the EBRT field. With a median follow-up of 36.1 months, 6 patients have relapsed, 2 of whom have died. Median progression-free survival was 17.5 months. Conclusions: In contrast to prior failure analysis data for RRBFL patients treated with {sup 90}Y-IT alone, a brief course of EBRT prevented relapse in sites of BD. EBRT used to pretreat bulky sites may improve clinical outcomes and potentially extend survival when combined with {sup 90}Y-IT.

  17. 2'-Deoxythymidine adducts from the anti-HIV drug nevirapine.

    PubMed

    Antunes, Alexandra M M; Wolf, Benjamin; Oliveira, M Conceição; Beland, Frederick A; Marques, M Matilde

    2013-04-26

    Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used against HIV-1. Currently, NVP is the most widely used anti-HIV drug in developing countries, both in combination therapy and to prevent mother-to-child transmission of HIV. Despite its efficacy against HIV, NVP produces a variety of toxic responses, including hepatotoxicity and skin rash. It is also associated with increased incidences of hepatoneoplasias in rodents. In addition, epidemiological data suggest that NNRTI use is a risk factor for non-AIDS-defining cancers in HIV-positive patients. Current evidence supports the involvement of metabolic activation to reactive electrophiles in NVP toxicity. NVP metabolism includes oxidation to 12-hydroxy-NVP; subsequent Phase II sulfonation produces an electrophilic metabolite, 12-sulfoxy-NVP, capable of reacting with DNA to yield covalent adducts. Since 2'-deoxythymidine (dT) adducts from several alkylating agents are regarded as having significant mutagenic/carcinogenic potential, we investigated the formation of NVP-dT adducts under biomimetic conditions. Toward this goal, we initially prepared and characterized synthetic NVP-dT adduct standards using a palladium-mediated Buchwald-Hartwig coupling strategy. The synthetic standards enabled the identification, by LC-ESI-MS, of 12-(2'-deoxythymidin-N3-yl)-nevirapine (N3-NVP-dT) in the enzymatic hydrolysate of salmon testis DNA reacted with 12-mesyloxy-NVP, a synthetic surrogate for 12-sulfoxy-NVP. N3-NVP-dT, a potentially cytotoxic and mutagenic DNA lesion, was also the only dT-specific adduct detected upon reaction of dT with 12-mesyloxy-NVP. Our data suggest that N3-NVP-dT may be formed in vivo and play a role in the hepatotoxicity and/or putative hepatocarcinogenicity of NVP.

  18. Quantification of Carnosine-Aldehyde Adducts in Human Urine.

    PubMed

    da Silva Bispo, Vanderson; Di Mascio, Paolo; Medeiros, Marisa

    2014-10-01

    Lipid peroxidation generates several reactive carbonyl species, including 4-hydroxy-2-nonenal (HNE), acrolein (ACR), 4-hydroxy-2-hexenal (HHE) and malondialdehyde. One major pathwayof aldehydes detoxification is through conjugation with glutathione catalyzed by glutathione-S-transferases or, alternatively, by conjugation with endogenous histidine containing dipeptides, such as carnosine (CAR). In this study, on-line reverse-phase high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection was utilized for the accurate quantification of CAR- ACR, CAR-HHE and CAR-HNE adducts in human urinary samples from non-smokers young adults. Standard adducts were prepared and isolated by HPLC. The results showed the presence of a new product from the reaction of CAR with ACR. This new adduct was completely characterized by HPLC/MS-MSn, 1H RMN, COSY and HSQC. The new HPLC/MS/MS methodology employing stable isotope-labeled internal standards (CAR-HHEd5 and CAR-HNEd11) was developed for adducts quantification. This methodology permits quantification of 10pmol CAR-HHE and 1pmol of CAR-ACR and CAR-HNE. Accurate determinations in human urine sample were performed and showed 4.65±1.71 to CAR-ACR, 5.13±1.76 to CAR-HHE and 5.99±3.19nmol/mg creatinine to CAR-HNE. Our results indicate that carnosine pathways can be an important detoxification route of a, ß -unsaturated aldehydes. Moreover, carnosine adducts may be useful as redox stress indicator. Copyright © 2014. Published by Elsevier Inc.

  19. Ion-molecule adduct formation in tandem mass spectrometry.

    PubMed

    Alechaga, Élida; Moyano, Encarnación; Galceran, Maria Teresa

    2016-02-01

    Nowadays most LC-MS methods rely on tandem mass spectrometry not only for quantitation and confirmation of compounds by multiple reaction monitoring (MRM), but also for the identification of unknowns from their product ion spectra. However, gas-phase reactions between charged and neutral species inside the mass analyzer can occur, yielding product ions at m/z values higher than that of the precursor ion, or at m/z values difficult to explain by logical losses, which complicate mass spectral interpretation. In this work, the formation of adduct ions in the mass analyzer was studied using several mass spectrometers with different mass analyzers (ion trap, triple quadrupole, and quadrupole-Orbitrap). Heterocyclic amines (AαC, MeAαC, Trp-P-1, and Trp-P-2), photo-initiators (BP and THBP), and pharmaceuticals (phenacetin and levamisole) were selected as model compounds and infused in LCQ Classic, TSQ Quantum Ultra AM, and Q-Exactive Orbitrap (ThermoFisher Scientific) mass spectrometers using electrospray as ionization method. The generation of ion-molecule adducts depended on the compound and also on the instrument employed. Adducts with neutral organic solvents (methanol and acetonitrile) were only observed in the ion trap instrument (LCQ Classic), because of the ionization source on-axis configuration and the lack of gas-phase barriers, which allowed inertial entrance of the neutrals into the analyzer. Adduct formation (only with water) in the triple quadrupole instruments was less abundant than in the ion trap and quadrupole-Orbitrap mass spectrometers, because of the lower residence time of the reactive product ions in the mass analyzer. The moisture level of the CID and/or damper gas had a great effect in beam-like mass analyzers such as triple quadrupole, but not in trap-like mass analyzers, probably because of the long residence time that allowed adduct formation even with very low concentrations of water inside the mass spectrometer.

  20. Parametric normalization for full-energy peak efficiency of HPGe γ-ray spectrometers at different counting positions for bulky sources.

    PubMed

    Peng, Nie; Bang-Fa, Ni; Wei-Zhi, Tian

    2013-02-01

    Application of effective interaction depth (EID) principle for parametric normalization of full energy peak efficiencies at different counting positions, originally for quasi-point sources, has been extended to bulky sources (within ∅30 mm×40 mm) with arbitrary matrices. It is also proved that the EID function for quasi-point source can be directly used for cylindrical bulky sources (within ∅30 mm×40 mm) with the geometric center as effective point source for low atomic number (Z) and low density (D) media and high energy γ-rays. It is also found that in general EID for bulky sources is dependent upon Z and D of the medium and the energy of the γ-rays in question. In addition, the EID principle was theoretically verified by MCNP calculations.

  1. Supramolecular bulky phosphines comprising 1,3,5-triaza-7-phosphaadamantane and Zn(salphen)s: structural features and application in hydrosilylation catalysis.

    PubMed

    Anselmo, Daniele; Gramage-Doria, Rafael; Besset, Tatiana; Escárcega-Bobadilla, Martha V; Salassa, Giovanni; Escudero-Adán, Eduardo C; Martínez Belmonte, Marta; Martin, Eddy; Reek, Joost N H; Kleij, Arjan W

    2013-06-07

    The use of the commercially available, bifunctional phosphine 1,3,5-triaza-7-phosphaadamantane (abbreviated as PN3) in conjunction with a series of Zn(salphen) complexes leads to sterically encumbered phosphine ligands as a result of (reversible) coordinative Zn-N interactions. The solid state and solution phase behaviour of these supramolecular ligand systems have been investigated in detail and revealed their stoichiometries in the solid state observed by X-ray crystallography, and those determined in solution by NMR and UV-Vis spectroscopy. Also, upon application of these supramolecular bulky phosphines in hydrosilylation catalysis employing 1-hexene as a substrate, the catalysis data infer the presence of an active Rh species with two coordinated, bulky PN3/Zn(salphen) assembly units having a maximum of three Zn(salphen)s associated per PN3 scaffold, with an excess of bulky phosphines hardly affecting the overall activity.

  2. In situ generated bulky palladium hydride complexes as catalysts for the efficient isomerization of olefins. Selective transformation of terminal alkenes to 2-alkenes.

    PubMed

    Gauthier, Delphine; Lindhardt, Anders T; Olsen, Esben P K; Overgaard, Jacob; Skrydstrup, Troels

    2010-06-16

    Application of an in situ generated bulky palladium(II) hydride catalyst obtained from a 1:1:1 mixture of Pd(dba)(2), P(tBu)(3), and isobutyryl chloride provides an efficient protocol for the isomerization and migration of a variety of olefins. In addition to the isomerization of (Z)- to (E)-olefins, the conjugative migration of allylbenzenes, allyl ethers, and amines was effectively achieved in near-quantitative yields and with excellent functional group tolerance. Catalyst loadings in the range of 0.5-1.0 mol % were typically applied, but even loadings as low as 0.25 mol % could be achieved when the reactions were performed under neat conditions. More interestingly, the investigated catalyst proved to be selective for converting terminal alkenes to 2-alkenes. This one-carbon migration process for monosubstituted olefins provides an alternative catalyst, which bridges the gap between the allylation and propenylation/vinylation protocols. Several substrates, including homoallylic alcohols and amines, were selectively transformed into their corresponding 2-alkenes, and examples using enantiomerically enriched substrates provided products without epimerization at the allylic stereogenic carbon centers. Finally, some mechanistic investigations were undertaken to understand the nature of the active in situ generated Pd-H catalyst. These studies revealed that the catalytic system is highly dependent on the large steric demand of the P(tBu)(3) ligand. The use of an alternative ligand, cataCXium PinCy, also proved effective for generating an active catalyst, and it was demonstrated in some cases to display better selectivity for the one-carbon shifts of terminal olefins. A possible intermediate involved in the preparation of the active catalyst was characterized by its single-crystal X-ray structure, which revealed a monomeric tricoordinated palladium(II) acyl complex, bearing a chloride ligand.

  3. Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

    SciTech Connect

    Guliaev, Anton B.; Singer, B.; Hang, Bo

    2004-05-05

    Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA than that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study.

  4. A Monoanionic Arsenide Source: Decarbonylation of the 2‐Arsaethynolate Anion upon Reaction with Bulky Stannylenes

    PubMed Central

    Hinz, Alexander

    2016-01-01

    Abstract We report fundamental studies on the reactivity of the 2‐arsaethynolate anion (AsCO−), a species that has only recently become synthetically accessible. The reaction of AsCO− with the bulky stannylene Ter2Sn (Ter=2,6‐bis[2,4,6‐trimethylphenyl]phenyl) is described, which leads to the unexpected formation of a [Ter3Sn2As2]− cluster compound. On the reaction pathway to this cluster, several intermediates were identified and characterized. After the initial association of AsCO− to Ter2Sn, decarbonylation occurs to give an anion featuring monocoordinate arsenic, [Ter2SnAs]−. Both species are not stable under ambient conditions, and [Ter2SnAs]− rearranges to form [TerSnAsTer]−, an unprecedented anionic mixed Group 14/15 alkene analogue. PMID:27862768

  5. Simple modification of basic dyes with bulky & symmetric WCAs for improving their solubilities in organic solvents without color change

    NASA Astrophysics Data System (ADS)

    Kim, Jeong Yun; Hwang, Tae Gyu; Woo, Sung Wun; Lee, Jae Moon; Namgoong, Jin Woong; Yuk, Sim Bum; Chung, Sei-Won; Kim, Jae Pil

    2017-04-01

    A simple and easy solubility enhancement of basic dyes was performed with bulky and symmetric weakly coordinating anions (WCAs). The WCAs decreased the ionic character of the dyes by broadening the partial charge distribution and causing a screening effect on the ionic bonding. This new modification with WCAs has advantages in that it has no influence on the optical properties of the dyes. The solubilities of unmodified and modified dyes were tested in several organic solvents. X-ray powder diffraction patterns of the dyes were measured. Color films were prepared with the dyes and their color loci were analyzed to evaluate the optical properties. By the modification with WCAs, commercial basic dyes showed sufficient solubilities for be applied to various applications while preserving their superior optical properties.

  6. Simple modification of basic dyes with bulky &symmetric WCAs for improving their solubilities in organic solvents without color change.

    PubMed

    Kim, Jeong Yun; Hwang, Tae Gyu; Woo, Sung Wun; Lee, Jae Moon; Namgoong, Jin Woong; Yuk, Sim Bum; Chung, Sei-Won; Kim, Jae Pil

    2017-04-06

    A simple and easy solubility enhancement of basic dyes was performed with bulky and symmetric weakly coordinating anions (WCAs). The WCAs decreased the ionic character of the dyes by broadening the partial charge distribution and causing a screening effect on the ionic bonding. This new modification with WCAs has advantages in that it has no influence on the optical properties of the dyes. The solubilities of unmodified and modified dyes were tested in several organic solvents. X-ray powder diffraction patterns of the dyes were measured. Color films were prepared with the dyes and their color loci were analyzed to evaluate the optical properties. By the modification with WCAs, commercial basic dyes showed sufficient solubilities for be applied to various applications while preserving their superior optical properties.

  7. Simple modification of basic dyes with bulky & symmetric WCAs for improving their solubilities in organic solvents without color change

    PubMed Central

    Kim, Jeong Yun; Hwang, Tae Gyu; Woo, Sung Wun; Lee, Jae Moon; Namgoong, Jin Woong; Yuk, Sim Bum; Chung, Sei-won; Kim, Jae Pil

    2017-01-01

    A simple and easy solubility enhancement of basic dyes was performed with bulky and symmetric weakly coordinating anions (WCAs). The WCAs decreased the ionic character of the dyes by broadening the partial charge distribution and causing a screening effect on the ionic bonding. This new modification with WCAs has advantages in that it has no influence on the optical properties of the dyes. The solubilities of unmodified and modified dyes were tested in several organic solvents. X-ray powder diffraction patterns of the dyes were measured. Color films were prepared with the dyes and their color loci were analyzed to evaluate the optical properties. By the modification with WCAs, commercial basic dyes showed sufficient solubilities for be applied to various applications while preserving their superior optical properties. PMID:28383019

  8. Covalent Tethering and Residues with Bulky Hydrophobic Side Chains Enable Self-Assembly of Distinct Amyloid Structures.

    PubMed

    Ruiz, Jérémy; Boehringer, Régis; Grogg, Marcel; Raya, Jésus; Schirer, Alicia; Crucifix, Corinne; Hellwig, Petra; Schultz, Patrick; Torbeev, Vladimir

    2016-12-02

    Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41] fragment of human β2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological, and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies, when applied to disease-related peptides and proteins, will greatly help in the evaluation of the biological properties of structurally distinct amyloids.

  9. 32P-postlabeling DNA adduct assay: cigarette smoke-induced dna adducts in the respiratory and nonrespiratory rat tissues. Book chapter

    SciTech Connect

    Gupta, R.C.; Gairola, C.G.

    1990-01-01

    An analysis of the tissue DNA adducts in rats by the sensitive (32)p-postlabeling assay showed one to eight detectable DNA adducts in lung, trachea, larynx, heart and bladder of the sham controls. Chronic exposure of animals to mainstream cigarette smoke showed a remarkable enhancement of most adducts in the lung and heart DNA. Since cigarette smoke contains several thousand chemicals and a few dozen of them are known or potential carcinogens, the difference between the DNA adducts of nasal and the other tissues may reflect the diversity of reactive constituents and their differential absorption in different tissues. In comparison to the lung DNA adducts, the adducts in nasal DNA were less hydrophobic. Identity of the predominant adducts was further investigated by comparison with several reference DNA adducts from 10 PAH and aromatic amines. Since some of these chemicals are present in cigarette smoke, the results suggest that these constituents of cigarette smoke may not be directly responsible for formation of DNA adducts in the lung and heart of the smoke-exposed animals.

  10. Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers.

    PubMed Central

    Harris, C C; Vahakangas, K; Newman, M J; Trivers, G E; Shamsuddin, A; Sinopoli, N; Mann, D L; Wright, W E

    1985-01-01

    Coke oven workers are exposed to high levels of carcinogenic polycyclic aromatic hydrocarbons, including benzo[a]pyrene (B[a]P), and are at increased risk of lung cancer. Since B[a]P is enzymatically activated to 7 beta,8 alpha-dihydroxy(9 alpha, 10 alpha)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) that forms adducts with DNA, the presence of these adducts was measured in DNA from peripheral blood lymphocytes by synchronous fluorescence spectrophotometry and enzyme radioimmunoassay. Approximately two-thirds of the workers had detectable levels of B[a]PDE-DNA adducts. Antibodies to the DNA adducts were also found in the serum of 27% of the workers. B[a]PDE-DNA adducts were not detectable in lymphocytes and antibodies to the adducts were not detected in sera from a control group of nonsmoking laboratory workers. DNA adducts and/or antibodies to the adducts indicate exposure to B[a]P and its metabolic activation to the carcinogenic metabolite that covalently binds to and damages DNA. Detection of adducts and antibodies to them may also be useful as internal dosimeters of the pathobiological effective doses of chemical carcinogens. PMID:2413443

  11. Assessing disease extent in women with bulky or clinically evident metastatic cervical cancer: yield of pretreatment studies.

    PubMed

    Massad, L S; Calvello, C; Gilkey, S H; Abu-Rustum, N R

    2000-03-01

    The objective of this study was to determine the impact of pretreatment tests on staging and management for women with bulky or clinically evident metastatic cervical cancer. Demographics and findings of clinic pelvic examination (PE), examination under anesthesia (EUA), chest x ray (CXR), abdominopelvic computed tomography (CT), and intravenous urography (IVU) were reviewed for women with primary, untreated cervical cancers either > or =4 cm or with extracervical disease apparent on PE or CXR evaluated between July 1, 1994, and March 31, 1999. Stage was assigned according to standards of the International Federation of Gynecology and Obstetrics. In 133 women studied, tumor diameter averaged 4. 9 cm on PE and 5.7 cm at EUA (P = 0.0005). Of 92 women undergoing both PE and EUA, 30 (33%) had size discrepancies of more than 1 cm. Compared with EUA, PE had sensitivity, specificity, and positive and negative predictive values, respectively, of 65, 89, 79, and 81% for vaginal disease, 74, 91, 95, and 63% for parametrial disease, and 57, 90, 60, and 89% for sidewall fixation. CXR was abnormal in 5 (4%). IVU showed ureteral dilation in 20 (22%) of 90 women, while CT showed dilation in 34 (28%) of 123 women. CT also showed pelvic lymphadenopathy in 22 (18%) women and paraaortic lymphadenopathy in 12 (10%). Bladder biopsies showed malignancy in 8 (8%), including one transitional cell carcinoma of the bladder. Proctoscopy failed to reveal metastatic cervical cancer. CXR and EUA with cystoscopy are important to the accurate staging and treatment of bulky or clinically metastatic cervical cancers, while proctoscopy is rarely useful. CT predicts ureteral obstruction well, and its greater information yield may justify its higher cost compared with IVU. Copyright 2000 Academic Press.

  12. Multiple Cycles of Neoadjuvant Chemotherapy Associated With Poor Survival in Bulky Stage IIIC and IV Ovarian Cancer.

    PubMed

    Ren, Yulan; Shi, Tingyan; Jiang, Rong; Yin, Sheng; Wang, Pan; Zang, Rongyu

    2015-10-01

    The aim of this study was to determine the impact of neoadjuvant chemotherapy (NACT) on survival in patients with bulky stage IIIC and IV epithelial ovarian cancer. Between January 2009 and December 2012, 408 patients with ovarian cancer with extensive upper abdominal disease were reviewed. On the basis of the cycle number of NACT, patients were divided into 2 groups, namely, primary debulking surgery (PDS) group, which included the patients who received no more than 1 cycle of NACT; and neoadjuvant chemotherapy followed by interval debulking surgery (NACT-IDS) group, which included the patients who received more than 1 cycle of NACT. All patients underwent cytoreductive surgery with the goal of optimal outcome (≤1 cm). Progression-free survival and overall survival were evaluated. There was no difference in surgical outcomes between PDS and NACT-IDS group, which was evaluated with either complete cytoreduction (41/376, 10.9% vs 6/32, 18.8%) or optimal cytoreduction (201/376, 53.5% vs 18/32, 56.2%). The median survival was 43.0 and 27.3 months in the PDS group and NACT-IDS group, with an estimated 5-year survival of 36% and 31%, respectively (P = 0.032; hazard ratio, 0.59; 95% confidence interval, 0.36-0.95). Complete cytoreduction, without bowel mesenteric carcinomatosis, and no more than 1 cycle of NACT were associated with lengthened survival by the multivariate analysis (P = 0.012, 0.025, and 0.036, respectively). Neoadjuvant chemotherapy was associated with poor survival of patients with bulky stage IIIC and IV ovarian cancer. A well-designed randomized trial with a better quality control of surgical procedures is needed to confirm the results.

  13. Detection of Adriamycin-DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations.

    PubMed

    Coldwell, Kate E; Cutts, Suzanne M; Ognibene, Ted J; Henderson, Paul T; Phillips, Don R

    2008-09-01

    Limited sensitivity of existing assays has prevented investigation of whether Adriamycin-DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin-DNA adducts/10(4) bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin-DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [(14)C]Adriamycin-DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin-DNA adducts at clinically-relevant Adriamycin concentrations. [(14)C]Adriamycin treatment (25 nM) resulted in 4.4 +/- 1.0 adducts/10(7) bp ( approximately 1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin-DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.

  14. [Methodology of capillary electrophoresis-laser induced fluorescence immunoassay for highly sensitive detection of DNA adducts].

    PubMed

    Wang, Hailin; Zhang, Dapeng; Wang, Zhixin; Li, Tao; Feng, Feng; Wang, Chao; Gao, Haiyan

    2009-09-01

    DNA adducts is a very important class of biomarkers of human exposure to carcinogen, cancer risk assessment, and population susceptibility. There is a lack of a technology with a sufficient sensitivity to detect trace DNA adducts related to low environmental exposure levels. We attempt to develop a highly sensitive assay for the detection of DNA adducts by combining capillary electrophoresis-laser induced fluorescence (CE-LIF) with immunochemical recognition, or CE-LIF immunoassay. This review describes the recent research progress on CE-LIF instrument construction and the methodology of CE-LIF immunoassay for the detection of DNA adducts. The methodology study mainly involves the synthesis and characterization of the adduct containing DNA fluorescent probes, the interaction of DNA adducts and antibody, stabilization of trace DNA adducts-antibody complexes, and DNA-driven focusing.

  15. Bending and circularization of site-specific and stereoisomeric carcinogen-DNA adducts.

    PubMed

    Xu, R; Mao, B; Amin, S; Geacintov, N E

    1998-01-13

    molecules of different sizes and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends, which permit efficient cyclization, especially in the case of (+)-trans-[BP]-N2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N2-dG lesion in the 11-mer duplex 5'-d(CACAT[G*]TACAC).d(GTGTACATGTG) indicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the duplex on the 5'-side of the modified guanosine residue, which may be due to bending enhanced base pair opening and bending induced by the bulky carcinogen residue. The implications of base sequence-dependent flexibilities and conformational mobilities of anti-[BP]-N2-dG lesions on DNA replication and mutation are discussed.

  16. Elucidation of reaction scheme describing malondialdehyde-acetaldehyde-protein adduct formation.

    PubMed

    Tuma, D J; Kearley, M L; Thiele, G M; Worrall, S; Haver, A; Klassen, L W; Sorrell, M F

    2001-07-01

    Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed animals. Our previous studies have shown that MAA adducts are comprised of two distinct products. One adduct is composed of two molecules of malondialdehyde and one molecule of acetaldehyde and was identified as the 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group (MDHDC adduct). The other adduct is a 1:1 adduct of malondialdehyde and acetaldehyde and was identified as the 2-formyl-3-(alkylamino)butanal derivative of an amino group (FAAB adduct). In this study, information on the mechanism of MAA adduct formation was obtained, focusing on whether the FAAB adduct serves as a precursor for the MDHDC adduct. Upon the basis of chemical analysis and NMR spectroscopy, two initial reaction steps appear to be a prerequisite for MDHDC formation. One step involves the reaction of one molecule of malondialdehyde and one of acetaldehyde with an amino group of a protein to form the FAAB product, while the other step involves the generation of a malondialdehyde-enamine. It appears that generation of the MDHDC adduct requires the FAAB moiety to be transferred to the nitrogen of the MDA-enamine. For efficient reaction of FAAB with the enamine to take place, additional experiments indicated that these two intermediates likely must be in positions on the protein of close proximity to each other. Further studies showed that the incubation of liver proteins from ethanol-fed rats with MDA resulted in a marked generation of MDHDC adducts, indicating the presence of a pool of FAAB adducts in the liver of ethanol-fed animals. Overall, these findings show that MDHDC-protein adduct formation occurs via the reaction of the FAAB moiety with a malondialdehyde-enamine, and further suggest that a similar mechanism may be operative in vivo in the liver during prolonged ethanol consumption.

  17. Theoretical study of the hydroxyl radical addition to uracil and photochemistry of the formed U6OH• adduct.

    PubMed

    Francés-Monerris, Antonio; Merchán, Manuela; Roca-Sanjuán, Daniel

    2014-03-20

    Hydroxyl radical ((•)OH) is produced in biological systems by external or endogenous agents. It can damage DNA/RNA by attacking pyrimidine nucleobases through the addition to the C5═C6 double bond. The adduct resulting from the attachment at the C5 position prevails in the experimental measurements, although the reasons for this preference remain unclear. The first aim of this work is therefore to shed light on the comprehension of this important process. Thus, the thermal (•)OH addition to the C5═C6 double bond of uracil has been studied theoretically by using DFT, MP2, and the multiconfigurational CASPT2//CASSCF methodologies. The in-vacuo results obtained with the latter protocol plus the analysis of solvent effects support the experimental observation. A significant lower barrier height is predicted for the C5 pathway with respect to that of the C6 route. In contrast to the C5 adduct, the C6 adduct is able to absorb visible light. Hence, the second aim of the work is to study the photochemistry of this species using the CASPT2//CASSCF methodology within the framework of the photochemical reaction path approach (PRPA). The nonradiative decay to the ground state of this compound has been characterized. A photoreactive character is predicted for the C6 adduct in the excited states according to the presence of excited-state minima along the main decay channel. Finally, a new mechanism of photodissociation has been explored, which implies the photoinduced regeneration of the canonical nucleobase by irradiating with visible light, being therefore relevant in RNA protection against damage by reactive oxygen species.

  18. Use of haemoglobin adducts in exposure monitoring and risk assessment.

    PubMed

    Boogaard, Peter J

    2002-10-05

    Many industrial bulk chemicals are oxiranes or alkenes that are easily metabolised to oxiranes in mammalian systems. Many oxiranes may react with DNA and are therefore mutagenic in vitro. Some oxiranes have been shown to be carcinogenic in rodents in vivo as well. Despite the very limited evidence of the carcinogenicity of oxiranes in humans, they should be considered potential human carcinogens. As a consequence, exposure to these compounds should be minimised and controlled. Twenty-five years ago, Ehrenberg and co-workers suggested that exposure to oxiranes might be determined through the measurement of the adducts they form with haemoglobin (Hb). Ten years later, a modification of the Edman degradation was developed at Stockholm University that allowed determination of adducts with the N-terminal valine of Hb by GC-MS. In our laboratory, this methodology was modified and adapted for analysis on an industrial scale. Since 1987, exposure of operators in our facilities to ethylene oxide (EO) has been routinely monitored by determination of N-(2-hydroxyethyl)valine in Hb. Biological monitoring programmes for propylene oxide (PO) and 1,3-butadiene (BD) were developed later. In this review, the methodology and its results are discussed as a tool in human risk assessment of industrial chemicals. Two major advantages of Hb adduct determinations in risk assessment are (1) the qualitative information on the structure of reactive intermediates that may be obtained through the mass spectrometry, which may provide insight in the molecular toxicology of compounds such as BD, and (2) the possibility of reliable determination of exposure over periods of several months with limited number of samples for compounds such as ethylene oxide (EO), propylene oxide (PO) and BD which form stable adducts with Hb. Since good correlations between the airborne concentrations of these chemicals with their respective adducts have been established, Hb adducts can also be used to quantitate

  19. UVR exposure sensitizes keratinocytes to DNA adduct formation.

    PubMed

    Nair, Sudhir; Kekatpure, Vikram D; Judson, Benjamin L; Rifkind, Arleen B; Granstein, Richard D; Boyle, Jay O; Subbaramaiah, Kotha; Guttenplan, Joseph B; Dannenberg, Andrew J

    2009-10-01

    UV radiation (UVR) and exposure to tobacco smoke, a source of polycyclic aromatic hydrocarbons (PAH), have been linked to skin carcinogenesis. UVR-mediated activation of the aryl hydrocarbon receptor (AhR) stimulates the transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAH to genotoxic metabolites. We determined whether UVR exposure sensitized human keratinocytes to PAH-induced DNA adduct formation. UVR exposure induced CYP1A1 and CYP1B1 in HaCaT cells, an effect that was mimicked by photooxidized tryptophan (aTRP) and FICZ, a component of aTRP. UVR exposure or pretreatment with aTRP or FICZ also sensitized cells to benzo(a)pyrene (B[a]P)-induced DNA adduct formation. alphaNF, an AhR antagonist, suppressed UVR-, aTRP-, and FICZ-mediated induction of CYP1A1 and CYP1B1 and inhibited B[a]P-induced DNA adduct formation. Treatment with 17-AAG, an Hsp90 inhibitor, caused a marked decrease in levels of AhR; inhibited UVR-, aTRP-, and FICZ-mediated induction of CYP1A1 and CYP1B1; and blocked the sensitization of HaCaT cells to B[a]P-induced DNA adduct formation. FICZ has been suggested to be a physiologic ligand of the AhR that may have systemic effects. Hence, studies of FICZ were also carried out in MSK-Leuk1 cells, a model of oral leukoplakia. Pretreatment with alpha-naphthoflavone or 17-AAG blocked FICZ-mediated induction of CYP1A1 and CYP1B1, and suppressed the increased B[a]P-induced DNA adduct formation. Collectively, these results suggest that sunlight may activate AhR signaling and thereby sensitize cells to PAH-mediated DNA adduct formation. Antagonists of AhR signaling may have a role in the chemoprevention of photocarcinogenesis.

  20. Adducts of mitomycin C and DNA in EMT6 mouse mammary tumor cells: effects of hypoxia and dicumarol on adduct patterns.

    PubMed

    Bizanek, R; Chowdary, D; Arai, H; Kasai, M; Hughes, C S; Sartorelli, A C; Rockwell, S; Tomasz, M

    1993-11-01

    6-CH3-3H-Mitomycin C (MC) was used to identify MC-DNA adducts formed in EMT6 mouse mammary tumor cells. DNA was isolated from cells treated with 3H-MC. The DNA was enzymatically digested, and the digest was analyzed for 3H-labeled adducts by high performance liquid chromatography. All four major adducts previously isolated and characterized in cell-free systems were detected: two different monoadducts and two bisadducts forming DNA-interstrand and DNA-intrastrand cross-links, respectively. No MC-DNA adducts other than the DNA interstrand cross-link had been shown previously to be formed in living cells. A MC-deoxyguanosine adduct of unknown structure was also detected in DNA from EMT6 cells; this adduct was also formed with purified EMT6 DNA. High performance liquid chromatography analysis was further applied to study the relationship between DNA adducts and cytotoxicity. The number of adducts increased with the concentration of MC in both aerobic and hypoxic cells. At a constant drug level, more adducts were observed in cells treated under hypoxic conditions than in cells treated aerobically; at 2 microM MC, 4.8 x 10(-7) and 3.1 x 10(-7) adducts/nucleotide were observed under hypoxic and aerobic conditions, respectively. The increased adduct frequency under hypoxia correlates with the known increased cytotoxicity of MC to EMT6 cells under hypoxic conditions. In addition, a higher ratio of cross-linked adducts to monoadducts was observed in hypoxic cells. The high performance liquid chromatography techniques were also used to examine the effects of dicumarol (DIC) on adduct patterns in cells treated simultaneously with 3H-MC. The MC-DNA adduct frequencies in DIC-treated cells were increased 1.5-fold under hypoxia and decreased 1.6-fold under aerobic conditions from those observed without DIC. This finding correlates with the known DIC-induced increase and decrease in the cytotoxicity of MC in hypoxic and aerobic EMT6 cells, respectively. The monoadduct resulting

  1. Biocidal properties of metal oxide nanoparticles and their halogen adducts

    NASA Astrophysics Data System (ADS)

    Haggstrom, Johanna A.; Klabunde, Kenneth J.; Marchin, George L.

    2010-03-01

    Nanosized metal oxide halogen adducts possess high surface reactivities due to their unique surface morphologies. These adducts have been used as reactive materials against vegetative cells, such as Escherichia coli as well as bacterial endospores, including Bacillus subtilis and Bacillus anthracis (Δ Sterne strain). Here we report high biocidal activities against gram-positive bacteria, gram-negative bacteria, and endospores. The procedure consists of a membrane method. Transmission electron micrographs are used to compare nanoparticle-treated and untreated cells and spores. It is proposed that the abrasive character of the particles, the oxidative power of the halogens/interhalogens, and the electrostatic attraction between the metal oxides and the biological material are responsible for high biocidal activities. While some activity was demonstrated, bacterial endospores were more resistant to nanoparticle treatment than the vegetative bacteria.

  2. Proteomic analysis of adducted butyrylcholinesterase for biomonitoring organophosphorus exposures

    PubMed Central

    Marsillach, Judit; Hsieh, Edward J.; Richter, Rebecca J.; MacCoss, Michael J.; Furlong, Clement E.

    2014-01-01

    Organophosphorus (OP) compounds include a broad group of toxic chemicals such as insecticides, chemical warfare agents and antiwear agents. The liver cytochromes P450 bioactivate many OPs to potent inhibitors of serine hydrolases. Cholinesterases were the first OP targets discovered and are the most studied. They are used to monitor human exposures to OP compounds. However, the assay that is currently used has limitations. The mechanism of action of OP compounds is the inhibition of serine hydrolases by covalently modifying their active-site serine. After structural rearrangement, the complex OP inhibitor-enzyme is irreversible and will remain in circulation until the modified enzyme is degraded. Mass spectrometry is a sensitive technology for analyzing protein modifications, such as OP-adducted enzymes. These analyses also provide some information about the nature of the OP adduct. Our aim is to develop high-throughput protocols for monitoring OP exposures using mass spectrometry. PMID:23123252

  3. Highly bulky and stable geometry-constrained iminopyridines: Synthesis, structure and application in Pd-catalyzed Suzuki coupling of aryl chlorides

    PubMed Central

    Lai, Yi; Zong, Zhijian; Tang, Yujie; Mo, Weimin; Sun, Nan; Hu, Baoxiang; Shen, Zhenlu

    2017-01-01

    A series of bulky geometry-constrained iminopyridylpalladium chlorides were developed. The steric environment adjacent to the nitrogen atom in the pyridine rings and diimine parts enhanced the thermal stability of the palladium species. Bulkier groups at the imino group stabilized the palladium species and the corresponding palladium chlorides showed high activities in the coupling reaction of aryl chlorides. PMID:28228862

  4. Structural and EPR characterisation of single electron and alkyl transfer products from reaction of dimethyl magnesium with bulky alpha-diimine ligands.

    PubMed

    Bailey, Philip J; Coxall, Robert A; Dick, Caroline M; Fabre, Sylvie; Parsons, Simon; Yellowlees, Lesley J

    2005-09-28

    Treatment of dimethylmagnesium with bulky alpha-diimine ligands provides either the biradical methyl-bridged complexes [(alpha-diimine-.)Mg+(mu-CH3)]2 via single electron transfer (SET), or the product of methyl transfer to an imine carbon atom depending upon conditions.

  5. Finite Element Biomechanics of Optic Nerve Sheath Traction in Adduction.

    PubMed

    Shin, Andrew; Yoo, Lawrence; Park, Joseph; Demer, Joseph L

    2017-10-01

    Historical emphasis on increased intraocular pressure (IOP) in the pathogenesis of glaucoma has been challenged by the recognition that many patients lack abnormally elevated IOP. We employed finite element analysis (FEA) to infer contribution to optic neuropathy from tractional deformation of the optic nerve head (ONH) and lamina cribrosa (LC) by extraocular muscle (EOM) counterforce exerted when optic nerve (ON) redundancy becomes exhausted in adduction. We characterized assumed isotropic Young's modulus of fresh adult bovine ON, ON sheath, and peripapillary and peripheral sclera by tensile elongation in arbitrary orientations of five specimens of each tissue to failure under physiological temperature and humidity. Physical dimensions of the FEA were scaled to human histological and magnetic resonance imaging (MRI) data and used to predict stress and strain during adduction 6 deg beyond ON straightening at multiple levels of IOP. Young's modulus of ON sheath of 44.6 ± 5.6 MPa (standard error of mean) greatly exceeded that of ON at 5.2 ± 0.4 MPa, peripapillary sclera at 5.5 ± 0.8 MPa, and peripheral sclera at 14.0 ± 2.3 MPa. FEA indicated that adduction induced maximum stress and strain in the temporal ONH. In the temporal LC, the maximum stress was 180 kPa, and the maximum strain was ninefold larger than produced by IOP elevation to 45 mm Hg. The simulation suggests that ON sheath traction by adduction concentrates far greater mechanical stress and strain in the ONH region than does elevated IOP, supporting the novel concept that glaucomatous optic neuropathy may result at least partly from external traction on the ON, rather than exclusively on pressure on the ON exerted from within the eye.

  6. Ion Pairs or Neutral Molecule Adducts? Cooperativity in Hydrogen Bonding

    ERIC Educational Resources Information Center

    DeKock, Roger L.; Schipper, Laura A.; Dykhouse, Stephanie C.; Heeringa, Lee P.; Brandsen, Benjamin M.

    2009-01-01

    We performed theoretical studies on the systems NH[subscript 3] times HF times mH[subscript 2]O, NH[subscript 3] times HCl times mH[subscript 2]O, with m = 0, 1, 2, and 6. The molecules with m = 0 form hydrogen-bonded adducts with little tendency to form an ion-pair structure. The molecule NH[subscript 3] times HCl times H[subscript 2]O cannot be…

  7. Ion Pairs or Neutral Molecule Adducts? Cooperativity in Hydrogen Bonding

    ERIC Educational Resources Information Center

    DeKock, Roger L.; Schipper, Laura A.; Dykhouse, Stephanie C.; Heeringa, Lee P.; Brandsen, Benjamin M.

    2009-01-01

    We performed theoretical studies on the systems NH[subscript 3] times HF times mH[subscript 2]O, NH[subscript 3] times HCl times mH[subscript 2]O, with m = 0, 1, 2, and 6. The molecules with m = 0 form hydrogen-bonded adducts with little tendency to form an ion-pair structure. The molecule NH[subscript 3] times HCl times H[subscript 2]O cannot be…

  8. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    SciTech Connect

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  9. 2' and 3' Carboranyl uridines and their diethyl ether adducts

    DOEpatents

    Soloway, Albert H.; Barth, Rolf F.; Anisuzzaman, Abul K.; Alam, Fazlul; Tjarks, Werner

    1992-01-01

    There is disclosed a process for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts, which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. Said carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of said compounds in methods for boron neutron capture therapy in mammalian tumor cells.

  10. Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a]pyrene dG adduct.

    PubMed

    Liu, Yang; Yang, Yeran; Tang, Tie-Shan; Zhang, Hui; Wang, Zhifeng; Friedberg, Errol; Yang, Wei; Guo, Caixia

    2014-02-04

    DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N(2)-deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N(2)-dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ(52-516)) leads to reduced polymerization activity, and the mutant PGM2(52-516) but not PGM1(52-516) can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ(52-516) retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.

  11. In Vivo Effects of Ozone Exposure on Protein Adduct Formation by 1-Nitronaphthalene in Rat Lung

    PubMed Central

    Wheelock, Åsa M.; Boland, Bridget C.; Isbell, Margaret; Morin, Dexter; Wegesser, Teresa C.; Plopper, Charles G.; Buckpitt, Alan R.

    2005-01-01

    The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36–0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone. PMID:15845863

  12. In vivo effects of ozone exposure on protein adduct formation by 1-nitronaphthalene in rat lung.

    PubMed

    Wheelock, Asa M; Boland, Bridget C; Isbell, Margaret; Morin, Dexter; Wegesser, Teresa C; Plopper, Charles G; Buckpitt, Alan R

    2005-08-01

    The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36-0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone.

  13. Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by sup 32 P-postlabeling analysis

    SciTech Connect

    Jahnke, G.D.; Thompson, C.L.; Walker, M.P.; Gallagher, J.E.; Lucier, G.W.

    1990-01-01

    Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. In the study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease {sup 32}P-post-labeling technique. Thin layer chromatography (TLC) maps from both groups revealed multiple DNA adducts which ranged from no adducts for one individual to six adducts for a different individual. The total DNA adduct concentrations were approximately one adduct in 10 to the seventh-10 to the eighth power normal nucleotides. Comparison of the adduct TLC profiles revealed individual variation in both pattern and level of DNA adducts. The type and amount of adduct was not influenced by smoking history and remained unchanged in four out of six subjects who were resampled after a one month interval. One adduct detected in lymphocyte DNA co-migrated on TLC with an adduct derived by in vitro incubation of lymphocytes with benzo(a)pyrene (B(a)P). The 3H-nucloside values were consistent with values obtained by {sup 32}P-postlabeling of the same sample (correlation coefficient of 0.88). No relationship was apparent between the capacity of lymphocytes to form a (3H)-B(a)P-derived adduct in vitro and the concentration of the adduct, or total adducts present in untreated lymphocytes.

  14. Tunable degradation of maleimide-thiol adducts in reducing environments

    PubMed Central

    Baldwin, Aaron D.; Kiick, Kristi L.

    2011-01-01

    Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here we show that the resulting succinimide thioether formed by a Michael type addition of a thiol to N-ethylmaleimide (NEM), generally accepted as stable, can in fact undergo retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies (1H NMR, HPLC) of NEM conjugated to 4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or 3-mercaptopropionic acid (MP) incubated with glutathione showed half lives of conversion from 20–80 hrs, with extents of conversion from 20–90% for MPA and N-acetylcysteine conjugates. Ring-opened the resultant succinimide thioether as well as any MP adduct did not show retro and exchange reactions. The kinetics of the retro reactions can be modulated by the Michael donor’s reactivity; therefore the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at timescales different than those currently possible via disulfide-mediated release. Such approaches may find a new niche for controlled release in reducing environments relevant in chemotherapy and sub-cellular trafficking. PMID:21863904

  15. Malondialdehyde-acetaldehyde adducts decrease bronchial epithelial wound repair.

    PubMed

    Wyatt, Todd A; Kharbanda, Kusum K; Tuma, Dean J; Sisson, Joseph H; Spurzem, John R

    2005-05-01

    Most people who abuse alcohol are cigarette smokers. Previously, we have shown that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, a component of both ethanol metabolism and cigarette smoke, form protein adducts that stimulate protein kinase C (PKC) activation in bronchial epithelial cells. We have also shown that PKC can regulate bronchial epithelial cell wound repair. We hypothesize that bovine serum albumin adducted with malondialdehyde and acetaldehyde (BSA-MAA) decreases bronchial epithelial cell wound repair via binding to scavenger receptors on bronchial epithelial cells. To test this, confluent monolayers of bovine bronchial epithelial cells were grown in serum-free media prior to wounding the cells. Bronchial epithelial cell wound closure was inhibited in a dose-dependent manner (up to 60%) in the presence of BSA-MAA than in media treated cells (Laboratory of Human Carcinogenesis [LHC]-9-Roswell Park Memorial Institute [RPMI]). The specific scavenger receptor ligand, fucoidan, also stimulated PKC activation and decreased wound repair. Pretreatment with fucoidan blocked malondialdehyde-acetaldehyde binding to bronchial epithelial cells. When bronchial epithelial cells were preincubated with a PKC alpha inhibitor, Gö 6976, the inhibition of wound closure by fucoidan and BSA-MAA was blocked. Western blot demonstrated the presence of several scavenger receptors on bronchial epithelial cell membranes, including SRA, SRBI, SRBII, and CD36. Scavenger receptor-mediated activation of PKC alpha may function to reduce wound healing under conditions of alcohol and cigarette smoke exposure where malondialdehyde-acetaldehyde adducts may be present.

  16. Thermal stability of DNA adducts induced by cyanomorpholinoadriamycin in vitro.

    PubMed Central

    Cullinane, C; Phillips, D R

    1993-01-01

    The Adriamycin derivative, cyanomorpholinoadriamycin (CMA) was reacted with DNA in vitro to form apparent interstrand crosslinks. The extent of interstrand crosslink formation was monitored by a gel electrophoresis assay and maximal crosslinking of DNA was observed within 1 hr with 5 microM of drug. The interstrand crosslinks were heat labile, with a midpoint melting temperature of 70 degrees C (10 min exposure to heat) in 45% formamide. When CMA-induced adducts were detected as blockages of lambda-exonuclease, 12 blockage sites were observed with 8 being prior to 5'-GG sequences, one prior to 5'-CC, one prior to 5'-GC and 2 at unresolved combinations of these sequences. These exonuclease-detected blockages reveal the same sites of CMA-induced crosslinking as detected by in vitro transcription footprinting and primer-extension blockages on single strand DNA, where the blockages at 5'-GG and 5'-CC were identified as sites of intrastrand crosslinking and the 5'-GC blockage as a probable site of interstrand crosslinking. The thermal stability of both types of crosslink (10 min exposure to heat) ranged from 63-70 degrees C at individual sites. High levels of adduct were detected with poly (dG-dC) but not with poly (dI-dC). These results suggest adduct formation involving an aminal linkage between the 3 position of the morpholino moiety and N2 of guanine. Images PMID:8493102

  17. Structural Characterization of Hydroxyl Radical Adducts in Aqueous Media

    NASA Astrophysics Data System (ADS)

    Janik, Ireneusz; Tripathi, G. N. R.

    2015-06-01

    The oxidation by the hydroxyl (OH) radical is one of the most widely studied reactions because of its central role in chemistry, biology, organic synthesis, and photocatalysis in aqueous environments, wastewater treatment, and numerous other chemical processes. Although the redox potential of OH is very high, direct electron transfer (ET) is rarely observed. If it happens, it mostly proceeds through the formation of elusive OH adduct intermediate which facilitates ET and formation of hydroxide anion. Using time resolved resonance Raman technique we structurally characterized variety of OH adducts to sulfur containing organic compounds, halide ions as well as some metal cations. The bond between oxygen of OH radical and the atom of oxidized molecule differs depending on the nature of solute that OH radical reacts with. For most of sulfur containing organics, as well as halide and pseudo-halide ions, our observation suggested that this bond has two-center three-electron character. For several metal aqua ions studied, the nature of the bond depends on type of the cation being oxidized. Discussion on spectral parameters of all studied hydroxyl radical adducts as well as the role solvent plays in their stabilization will be presented.

  18. Isolation of Cyclopropenylidene Lithium Adducts: The Weiss-Yoshida Reagent**

    PubMed Central

    Lavallo, Vincent; Ishida, Yutaka; Donnadieu, Bruno; Bertrand, Guy

    2008-01-01

    A lithium-halogen exchange reaction occurs when the chloro[bis(diisopropylamino)]cyclopropenium tetrafluoroborate salt 1 (X = BF4) is treated with n-butyllithium. The resulting cyclopropenylidene-lithium adduct 3 has been isolated in 45% yield. In the solid state, this compound exists as a polymeric chain with an overall stoichiometry of two LiBF4 per carbene ligand. Addition of 12-crown-4-ether does not liberate the carbene from the lithium cation, but affords a monomeric tertiary complex (60% yield) that includes the crown ether. Moreover, complex 3 can also be synthesized by depro tonation of the bis(diisopropylamino)cyclopropenium tetrafluoroborate salt 2 (X = BF4) with n-butyllithium, whereas using potassium bis(trimethylsilyl)amide the free cyclopropenylidene was isolated in 53% yield. These results as whole seem to demonstrate that only certain counteranions allow for the isolation of cyclopropenylidene-lithium adducts, and only bases not containing lithium allow for the isolation of the free cyclopropenylidene. The former and the latter presumably prevented Weiss and Yoshida from isolating what would have been the first example of a stable carbene-lithium adduct and a free carbene, respectively. PMID:16986195

  19. Detection and characterization of cyclic hydroxylamine adducts by mass spectrometry.

    PubMed

    Reis, Ana; Domingues, Maria R M; Amado, Francisco M L; Oliveira, M Manuel; Domingues, Pedro

    2008-05-01

    Two cyclic hydroxylamines (cHA) bearing pyrrolidine (CPH) and piperidine moieties (TMTH) were evaluated to trap hydroxyl, peptide and phospholipid free radicals using mass spectrometry for their detection. The cHA ionized as [M+H](+) ions, showing higher relative abundances when compared to the DMPO, probably due to higher ionization efficiency. In the presence of hydroxyl radicals, both cHA generated new ions that could be attributed to loss of (*)H and (*)CH(3), most likely deriving from decomposition reactions of the nitroxide spin adduct. Addition of cHA to Leucine-enkephalin and palmitoyl-lineloyl-glycerophosphatidylcholine free radicals promoted the formation of cHA biomolecule adducts, which were confirmed by MS/MS data. Results suggest that the cHA are not suitable for hydroxyl radical trapping but can be used for trapping biomolecule radicals, having the advantage, over the most used cyclic nitrones, of being water soluble. The biomolecule adducts identified by MS are ESR silent, evidencing the importance of MS detection.

  20. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    PubMed Central

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. PMID:23566956

  1. Immunochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes.

    PubMed

    Uchida, K; Szweda, L I; Chae, H Z; Stadtman, E R

    1993-09-15

    We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.

  2. Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA adduct formation

    PubMed Central

    Russell, Gilandra K.; Gupta, Ramesh C.; Vadhanam, Manicka V.

    2015-01-01

    Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the environment are combustion of wood and coal burning, gasoline and diesel exhaust, and tires. Given the likelihood of environmental exposure to DBP and strong experimental evidence of its potency, it is likely to contribute to lung cancer development. Intervention with compounds of natural origin (“phytochemicals”) is considered an effective means to prevent cancer development and favorably modulate the underlying mechanisms, including DNA adduct formation. In this study, several agents have been identified that inhibit environmental carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of the ten agents tested, resveratrol (648 ± 26 adducts/109 nucleotides), oltipraz (1007 ± 348 adducts/109 nucleotides), delphinidin (1252 ± 142 adducts/109 nucleotides), tanshinone I (1981 ± 213 adducts/109 nucleotides), tanshinone IIA (2606 ± 478 adducts/109 nucleotides) and diindoylmethane (3643 ± 469 adducts/109 nucleotides) were the most effective compared to vehicle treatment (14,062 ± 1097 adducts/109 nucleotides). DBP is metabolized by phase I metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be inhibited by several mechanisms. We found that all the test agents inhibited DNA adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin inhibited DNA adducts by also interacting directly with the carcinogenic metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide. PMID:25794985

  3. Acetaminophen Adducts Detected in Serum of Pediatric Patients With Acute Liver Failure.

    PubMed

    Alonso, Estella M; James, Laura P; Zhang, Song; Squires, Robert H

    2015-07-01

    Previous studies in patients with acute liver failure identified acetaminophen (APAP) protein adducts in the serum of 12% and 19% of children and adults, respectively, with acute liver failure of indeterminate etiology. This article details the testing of APAP adducts in a subset (n = 393) of patients with varied diagnoses in the Pediatric Acute Liver Failure Study Group (PALFSG). Serum samples were available from 393 participants included in the PALFSG registry. Adduct measurement was performed using validated methods. Participants were grouped by diagnostic category as known APAP overdose, known other diagnosis, and indeterminate etiology. Demographic and clinical characteristics and participant outcomes were compared by adduct status (positive or negative) within each group. APAP adduct testing was positive in 86% of participants with known APAP overdose, 6% with other known diagnoses, and 11% with an indeterminate cause of liver failure. Adduct-positive participants were noted to have marked elevation of serum alanine aminotransferase and aspartate aminotransferase coupled with total serum bilirubin that was significantly lower than adduct-negative patients. In the indeterminate group, adduct-positive patients had different outcomes than adduct-negative patients (P = 0.03); spontaneous survival was 16 of 21 (76%) in adduct-positive patients versus 75 of 169 (44%) in adduct-negative patients. Prognosis did not vary by adduct status in patients with known diagnoses. Furthermore, study is needed to understand the relation of APAP exposure, as determined by the presence of APAP adducts, to the clinical phenotype and outcomes of children with acute liver failure.

  4. Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA adduct formation.

    PubMed

    Russell, Gilandra K; Gupta, Ramesh C; Vadhanam, Manicka V

    2015-04-01

    Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the environment are combustion of wood and coal burning, gasoline and diesel exhaust, and tires. Given the likelihood of environmental exposure to DBP and strong experimental evidence of its potency, it is likely to contribute to lung cancer development. Intervention with compounds of natural origin ("phytochemicals") is considered an effective means to prevent cancer development and favorably modulate the underlying mechanisms, including DNA adduct formation. In this study, several agents have been identified that inhibit environmental carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of the ten agents tested, resveratrol (648 ± 26 adducts/10(9) nucleotides), oltipraz (1007 ± 348 adducts/10(9) nucleotides), delphinidin (1252 ± 142 adducts/10(9) nucleotides), tanshinone I (1981 ± 213 adducts/10(9) nucleotides), tanshinone IIA (2606 ± 478 adducts/10(9) nucleotides) and diindoylmethane (3643 ± 469 adducts/10(9) nucleotides) were the most effective compared to vehicle treatment (14,062 ± 1097 adducts/10(9) nucleotides). DBP is metabolized by phase I metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be inhibited by several mechanisms. We found that all the test agents inhibited DNA adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin inhibited DNA adducts by also interacting directly with the carcinogenic metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Hip adduction and abduction strength profiles in elite, sub-elite and amateur Australian footballers.

    PubMed

    Prendergast, Ned; Hopper, Diana; Finucane, Mark; Grisbrook, Tiffany L

    2016-09-01

    It has been reported that obtaining an adduction-to-abduction strength ratio of 90-100%, and an adduction strength equal to that of the uninjured side, are suitable clinical milestones for return to sport following groin injury. Little is known about hip adduction and abduction strength profiles in Australian footballers. This study aimed to compare isometric hip adduction and abduction strength profiles between preferred and non-preferred kicking legs in elite, sub-elite and amateur Australian footballers. Cross sectional study 36 elite, 19 sub-elite and 18 amateur Australian footballers, with a mean age of 24, 19 and 23 years respectively, were included. Maximal hip isometric adduction and abduction strength were measured using a hand held dynamometer with external belt fixation. There were no significant differences in isometric hip adduction (p=0.262) or abduction (p=0.934) strength, or the adduction-to-abduction ratio (p=0.163), between preferred and non-preferred kicking legs, regardless of playing level. Elite players had significantly greater isometric hip adduction and abduction strength than both sub-elite (mean difference; adduction=46.01N, p<0.001, abduction=30.79N, p=0.003) and amateur players (mean difference; adduction=78.72N, p<0.001, abduction=59.11N, p<0.001). There was no significant difference in the adduction-to-abduction ratio between the playing levels (p=0.165). No significant differences were found between preferred and non-preferred kicking legs across the playing levels for isometric hip adduction, abduction or the adduction-to-abduction ratio. This may have implications for developing groin injury prediction and return to sport criteria in Australian footballers. Copyright © 2015 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  6. Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

    PubMed

    Tokarsky, E John; Gadkari, Varun V; Zahurancik, Walter J; Malik, Chanchal K; Basu, Ashis K; Suo, Zucai

    2016-10-01

    3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG(C8-)(N)(-ABA)), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dG(C8-)(N)(-ABA) can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dG(C8-)(N)(-ABA)in vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dG(C8-)(N-)(ABA) lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dG(C8-)(N)(-ABA). In contrast, hPolι and hRev1 were severely stalled by dG(C8-)(N)(-ABA) and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dG(C8-)(N-)(ABA). Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dG(C8-)(N)(-ABA) bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Effects of Bulky Substituents of Push-Pull Porphyrins on Photovoltaic Properties of Dye-Sensitized Solar Cells.

    PubMed

    Higashino, Tomohiro; Kawamoto, Kyosuke; Sugiura, Kenichi; Fujimori, Yamato; Tsuji, Yukihiro; Kurotobi, Kei; Ito, Seigo; Imahori, Hiroshi

    2016-06-22

    To evaluate the effects of substituent bulkiness around a porphyrin core on the photovoltaic properties of porphyrin-sensitized solar cells, long alkoxy groups were introduced at the meso-phenyl group (ZnPBAT-o-C8) and the anchoring group (ZnPBAT-o-C8Cn, n = 4, 8) of an asymmetrically substituted push-pull porphyrin with double electron-donating diarylamino groups and a single electron-withdrawing carboxyphenylethynyl anchoring group. The spectroscopic and electrochemical properties of ZnPBAT-o-C8 and ZnPBAT-o-C8Cn were found to be superior to those of a push-pull porphyrin reference (YD2-o-C8), demonstrating their excellent light-harvesting and redox properties for dye-sensitized solar cells. A power conversion efficiency (η) of the ZnPBAT-o-C8-sensitized solar cell (η = 9.1%) is higher than that of the YD2-o-C8-sensitized solar cell (η = 8.6%) using iodine-based electrolyte due to the enhanced light-harvesting ability of ZnPBAT-o-C8. In contrast, the solar cells based on ZnPBAT-o-C8Cn, possessing the additional alkoxy chains in the anchoring group, revealed the lower η values of 7.3% (n = 4) and 7.0% (n = 8). Although ZnPBAT-o-C8Cn exhibited higher resistance at the TiO2-dye-electrolyte interface by virtue of the extra alkoxy chains, the reduced amount of the porphyrins on TiO2 by excessive addition of coadsorbent chenodeoxycholic acid (CDCA) for mitigating the aggregation on TiO2 resulted in the low η values. Meanwhile, the ZnPBAT-o-C8-sensitized solar cell showed the lower η value of 8.1% than the YD2-o-C8-sensitized solar cell (η = 9.8%) using cobalt-based electrolyte. The smaller η value of the ZnPBAT-o-C8-sensitized solar cell may be attributed to the insufficient blocking effect of the bulky substituents of ZnPBAT-o-C8 under the cobalt-based electrolyte conditions. Overall, the alkoxy chain length and substitution position around the porphyrin core are important factors to affect the cell performance.

  8. Including the Copenhagen Adduction Exercise in the FIFA 11+ Provides Missing Eccentric Hip Adduction Strength Effect in Male Soccer Players: A Randomized Controlled Trial.

    PubMed

    Harøy, Joar; Thorborg, Kristian; Serner, Andreas; Bjørkheim, André; Rolstad, Linn E; Hölmich, Per; Bahr, Roald; Andersen, Thor Einar

    2017-08-01

    The FIFA 11+ was developed as a complete warm-up program to prevent injuries in soccer players. Although reduced hip adduction strength is associated with groin injuries, none of the exercises included in the FIFA 11+ seem to specifically target hip adduction strength. To investigate the effect on eccentric hip adduction strength of the FIFA 11+ warm-up program with or without the Copenhagen adduction exercise. Randomized controlled trial; Level of evidence, 1. We recruited 45 eligible players from 2 U19 elite male soccer teams. Players were randomized into 2 groups; 1 group carried out the standard FIFA 11+ program, while the other carried out the FIFA 11+ but replaced the Nordic hamstring exercise with the Copenhagen adduction exercise. Both groups performed the intervention 3 times weekly for 8 weeks. Players completed eccentric strength and sprint testing before and after the intervention. Per-protocol analyses were performed, and 12 players were excluded due to low compliance (<67% of sessions completed). The main outcome was eccentric hip adduction strength (N·m/kg). Between-group analyses revealed a significantly greater increase in eccentric hip adduction strength of 0.29 Nm/kg (8.9%; P = .01) in favor of the group performing the Copenhagen adduction exercise, whereas no within-group change was noted in the group that used the standard FIFA 11+ program (-0.02 N·m/kg [-0.7%]; P = .69). Including the Copenhagen adduction exercise in the FIFA 11+ program increases eccentric hip adduction strength, while the standard FIFA 11+ program does not. Registration: Registration: ISRCTN13731446 (International Standard Randomised Controlled Trial Number registry).

  9. Serological characterization of polycyclic aromatic hydrocarbon diolepoxide-DNA adducts using monoclonal antibodies.

    PubMed

    Newman, M J; Weston, A; Carver, D C; Mann, D L; Harris, C C

    1990-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide-DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH-DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH-DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus demonstrating their specificity for PAH-DNA adducts and not the PAHs alone

  10. Low response in white blood cell DNA adducts among workers in a highly polluted cokery environment.

    PubMed

    Kuljukka, T; Savela, K; Vaaranrinta, R; Mutanen, P; Veidebaum, T; Sorsa, M; Peltonen, K

    1998-06-01

    Coke oven workers are often heavily exposed to polynuclear aromatic hydrocarbons (PAHs); this exposure has been associated with higher cancer rates among these workers. We assessed the exposure of cokery workers in an oil shale processing plant. Personal hygienic monitoring, measurement of urinary 1-hydroxypyrene (1-OHP), and analysis of PAH-DNA adducts in white blood cells (WBCs) were performed. The 32P-postlabeling method was used for adduct measurement. The mean adduct value, 1.6 adducts per 10(8) nucleotides, did not differ significantly from the control value (P = 0.098). Smokers had significantly higher adduct levels than non-smoking workers (P = 0.002). 1-OHP levels measured in post-shift samples correlated with DNA adducts found in white blood cells (WBCs). We conclude that hygienic monitoring and measurement of urinary metabolites are essential background exposure data when the biologically effective dose of chemical carcinogens is assessed.

  11. Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

    PubMed Central

    Lindroth, S; von Wright, A

    1978-01-01

    The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg. PMID:354524

  12. Synthesis, characterization, and solvolysis of mono- and bis-S-(glutathionyl) adducts of methylene-bis-(phenylisocyanate) (MDI).

    PubMed

    Reisser, Martin; Schmidt, Brigitte F; Brown, William E

    2002-10-01

    degradation, a variety of products were formed upon degradation of the bis adduct. Though the disappearance of the bis adduct was complete as measured by HPLC, (1)H NMR spectra showed the existence of residual thiocarbamate bonds in the final mixture. In both cases, no evidence of the free methylene-bis-phenylamine (MDA) could be detected under the applicable conditions.

  13. Hydrolytic Cleavage Products of Globin Adducts in Urine as Possible Biomarkers of Cumulative Dose: Proof of Concept Using Styrene Oxide as a Model Adduct-Forming Compound.

    PubMed

    Mráz, Jaroslav; Hanzlíková, Iveta; Moulisová, Alena; Dušková, Šárka; Hejl, Kamil; Bednářová, Aneta; Dabrowská, Ludmila; Linhart, Igor

    2016-04-18

    A new experimental model was designed to study the fate of globin adducts with styrene 7,8-oxide (SO), a metabolic intermediate of styrene and a model electrophilic compound. Rat erythrocytes were incubated with SO at 7 or 22 °C. Levels of specific amino acid adducts in globin were determined by LC/MS analysis of the globin hydrolysate, and erythrocytes with known adduct content were administered intravenously to recipient rats. The course of adduct elimination from the rat blood was measured over the following 50 days. In the erythrocytes incubated at 22 °C, a rapid decline in the adduct levels on the first day post-transfusion followed by a slow phase of elimination was observed. In contrast, the adduct elimination in erythrocytes incubated at 7 °C was nearly linear, copying elimination of intact erythrocytes. In the urine of recipient rats, regioisomeric SO adducts at cysteine, valine, lysine, and histidine in the form of amino acid adducts and/or their acetylated metabolites as well as SO-dipeptide adducts were identified by LC/MS supported by synthesized reference standards. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine, the most abundant globin adducts, were excreted predominantly in the form of the corresponding urinary mercapturic acids (HPEMAs). Massive elimination of HPEMAs via urine occurred within the first day from the erythrocytes incubated at both 7 and 22 °C. However, erythrocytes incubated at 7 °C also showed a slow second phase of elimination such that HPEMAs were detected in urine up to 50 days post-transfusion. These results indicate for the first time that globin adducts can be cleaved in vivo to modified amino acids and dipeptides. The cleavage products and/or their predictable metabolites are excreted in urine over the whole life span of erythrocytes. Some of the urinary adducts may represent a new type of noninvasive biomarker for exposure to adduct-forming chemicals.

  14. Pendant Ancillary Ligand Switches Off Auto-Oxidation of Group 13 Metal Alkyl Compounds Bearing Non-Bulky Alkyl Groups

    PubMed Central

    Kobrsi, Issam

    2014-01-01

    The reaction of 3,5-di-2-pyridyl-1,2,4-triazole with excess Al(CH3)3 and Ga(CH3)3 afforded (3,5-di-2-pyridyl-1,2,4-triazolate)Al(CH3)2•3Al(CH3)3 (1) and (3,5-di-2-pyridyl-1,2,4-triazolate) Ga(CH3)2•3Ga(CH3)3 (2) respectively. 1 and 2 reacted with oxygen gas to produce (CH3)2M(µ-3,5-di-2-pyridyl-1,2,4-triazolate)(µ-OCH3)M(CH3)2 (M = Al, 3; M = Ga, 4). 3 and 4 contain the non-bulky dimethylalumino moiety, yet they are indefinitely stable in the presence of oxygen gas. This increased stability towards oxygen is due to ancillary 2-pyridyl groups bonding to the metal centers producing a pseudo-trigonal pyramidal Al and Ga environments. This environment blocks oxygen from further inserting into the M–C bond. The Al–N(pyridine) and Ga–N(pyridine) bonds reported herein are extremely elongated yet inactive towards dissociation due to the chelate effect. PMID:24945752

  15. Pendant ancillary ligand switches off auto-oxidation of group 13 metal alkyl compounds bearing non-bulky alkyl groups.

    PubMed

    Kobrsi, Issam

    2014-01-01

    The reaction of 3,5-di-2-pyridyl-1,2,4-triazole with excess Al(CH3)3 and Ga(CH3)3 afforded (3,5-di-2-pyridyl-1,2,4-triazolate)Al(CH3)2•3Al(CH3)3 (1) and (3,5-di-2-pyridyl-1,2,4-triazolate) Ga(CH3)2•3Ga(CH3)3 (2) respectively. 1 and 2 reacted with oxygen gas to produce (CH3)2M(µ-3,5-di-2-pyridyl-1,2,4-triazolate)(µ-OCH3)M(CH3)2 (M = Al, 3; M = Ga, 4). 3 and 4 contain the non-bulky dimethylalumino moiety, yet they are indefinitely stable in the presence of oxygen gas. This increased stability towards oxygen is due to ancillary 2-pyridyl groups bonding to the metal centers producing a pseudo-trigonal pyramidal Al and Ga environments. This environment blocks oxygen from further inserting into the M-C bond. The Al-N(pyridine) and Ga-N(pyridine) bonds reported herein are extremely elongated yet inactive towards dissociation due to the chelate effect.

  16. A phase I-II trial of multimodality management of bulky gynecologic malignancy. Combined chemoradiosensitization and radiotherapy

    SciTech Connect

    Kersh, C.R.; Constable, W.C.; Spaulding, C.A.; Hahn, S.S.; Andersen, W.A.; Taylor, P.T. )

    1990-07-01

    Between December 1983 and December 1987, there were 44 patients with bulky, nonresectable squamous cell carcinomas of the gynecologic tract (cervix, 36; vagina, eight) who were treated with concomitant chemotherapy and radiotherapy. Chemotherapy consisted of 5-fluorouracil (5-FU) 1g/m2 given by continuous intravenous infusion on days 1 through 4 and mitomycin C 10 mg/m2 given intravenously on day 1. External-beam irradiation was started on day 1 with a total calculated dose of 5000 cGy in 25 fractions employed. This was followed by brachytherapy. With a mean follow-up of 30.3 months and a median of 28 months, local control has been achieved in 32 of 44 patients (73%). The overall response rate was 88% (3-month partial response, 43%; 3-month complete response, 45%; 8-month partial response, 15%; 8-month complete response, 73%). Analysis of complications by Radiation Therapy Oncology Group (RTOG) criteria did not demonstrate an increase in acute or late complications.

  17. Assessment of the cryoprotectant concentration inside a bulky organ for cryopreservation using X-ray computed tomography.

    PubMed

    Corral, Ariadna; Balcerzyk, Marcin; Parrado-Gallego, Ángel; Fernández-Gómez, Isabel; Lamprea, David R; Olmo, Alberto; Risco, Ramón

    2015-12-01

    Cryoprotection of bulky organs is crucial for their storage and for subsequent transplantation. In this work we demonstrate the capability of the X-ray computed tomography (CT) as a non-invasive method to measure the cryoprotectant (cpa) concentration inside a tissue or an organ, specifically for the case of dymethil sulfoxide (Me2SO). It is remarkable that the use of Me2SO has been leader in techniques of cells and tissues cryopreservation. Although CT technologies are mainly based in density differences, and many cpas are alcohols with densities similar to water, the use of very low energies as acceleration voltage (∼70 kV) and the sulfur atom in the molecule of Me2SO makes possible the visualization of this cpa inside tissues. As result we obtain a CT signal proportional to the Me2SO concentration with a spatial resolution up to 50 μm in the case of our device. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Structure of adducts of isoindolo[2,1-a]benzimidazole derivatives with maleimides

    NASA Astrophysics Data System (ADS)

    Korolev, Oleksandr; Yegorova, Tatyana; Levkov, Igor; Malytskyy, Volodymyr; Shishkin, Oleg; Zubatyuk, Roman; Palamarchuk, Genadiy; Vedrenne, Marc; Baltas, Michel; Voitenko, Zoia

    2015-03-01

    The selectivity of formation and some mechanistic insights during the synthesis of substituted isoindolo[2,1-a]benzimidazoles are discussed. Furthermore, the reactions of the obtained products with maleimides were carried out. Two types rearrangement adducts together with intermediate Michael type adducts were isolated. The influence of the reaction conditions and reagents ratio is discussed. Specific spectral criteria for the identification of the Michael type adducts are indicated.

  19. Correlation between Quadriceps Endurance and Adduction Moment in Medial Knee Osteoarthritis

    PubMed Central

    Ahn, Sung-Eun; Park, Min-Ji; Lee, Dae-Hee

    2015-01-01

    It is not clear whether the strength or endurance of thigh muscles (quadriceps and hamstring) is positively or negatively correlated with the adduction moment of osteoarthritic knees. This study therefore assessed the relationships between the strength and endurance of the quadriceps and hamstring muscles and adduction moment in osteoarthritic knees and evaluated predictors of the adduction moment. The study cohort comprised 35 patients with unilateral medial osteoarthritis and varus deformity who were candidates for open wedge osteotomy. The maximal torque (60°/sec) and total work (180°/sec) of the quadriceps and hamstring muscles and knee adduction moment were evaluated using an isokinetic testing device and gait analysis system. The total work of the quadriceps (r = 0.429, P = 0.037) and hamstring (r = 0.426, P = 0.045) muscles at 180°/sec each correlated with knee adduction moment. Preoperative varus deformity was positively correlated with adduction moment (r = 0.421, P = 0.041). Multiple linear regression analysis showed that quadriceps endurance at 180°/sec was the only factor independently associated with adduction moment (β = 0.790, P = 0.032). The adduction moment of osteoarthritic knees correlated with the endurance, but not the strength, of the quadriceps muscle. However, knee adduction moment did not correlate with the strength or endurance of the hamstring muscle. PMID:26539830

  20. Lifetimes and stabilities of familiar explosives molecular adduct complexes during ion mobility measurements

    PubMed Central

    McKenzie, Alan; DeBord, John Daniel; Ridgeway, Mark; Park, Melvin; Eiceman, Gary; Fernandez-Lima, Francisco

    2015-01-01

    Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) was utilized for the separation and identification of familiar explosives in complex mixtures. For the first time, molecular adduct complex lifetimes, relative stability, binding energies and candidate structures are reported for familiar explosives. Experimental and theoretical results showed that the adduct size and reactivity, complex binding energy and the explosive structure tailors the stability of the molecular adduct complex. TIMS flexibility to adapt the mobility separation as a function of the molecular adduct complex stability (i.e., short or long IMS experiments / low or high IMS resolution) permits targeted measurements of explosives in complex mixtures with higher confidence levels. PMID:26153567

  1. Temporal and spatial features of the formation of DNA adducts in sulfur mustard-exposed skin

    SciTech Connect

    Batal, Mohamed; Boudry, Isabelle; Mouret, Stéphane; Wartelle, Julien; Emorine, Sandy; Bertoni, Marine; Bérard, Izabel; and others

    2013-12-15

    Sulfur mustard (SM) is a chemical warfare agent that targets skin where it induces large blisters. DNA alkylation is a critical step to explain SM-induced cutaneous symptoms. We determined the kinetics of formation of main SM–DNA adducts and compare it with the development of the SM-induced pathogenesis in skin. SKH-1 mice were exposed to 2, 6 and 60 mg/kg of SM and treated skin was biopsied between 6 h and 21 days. Formation of SM DNA adducts was dose-dependent with a maximum immediately after exposure. However, adducts were persistent and still detectable 21 days post-exposure. The time-dependent formation of DNA adducts was also found to be correlated with the appearance of apoptotic cells. This temporal correlation suggests that these two early events are responsible for the severity of the damage to the skin. Besides, SM–DNA adducts were also detected in areas located next to contaminated zone, thus suggesting that SM diffuses in skin. Altogether, this work provides for the first time a clear picture of SM-induced genotoxicity using DNA adducts as a marker. - Highlights: • Sulfur mustard adducts are formed in DNA after skin exposure. • DNA damage formation is an early event in the pathological process of skin burn. • The amount of SM–DNA adducts is maximal at the earliest time point investigated. • Adducts are still detected 3 weeks after exposure. • Sulfur mustard diffuses in skin especially when large doses are applied.

  2. Comparison of biological effects with albumin adducts of 4,4'-methylenediphenyl diisocyanate in workers.

    PubMed

    Sabbioni, Gabriele; Vanimireddy, Lakshiminiranjan Reddy; Lummus, Zana L; Bernstein, David I

    2017-04-01

    Lung sensitization and asthma are the main health effects of 4,4'-methylenediphenyl diisocyanate (MDI). Albumin adducts (isocyanate-specific adducts) of MDI might be involved in the etiology of sensitization reactions. Albumin adducts of MDI were analyzed in sera of diisocyanate-exposed worker with and without diisocyanate occupational asthma (DA), as well as in exposed workers with and without diisocyanate-specific IgG antibodies. In DA-positive workers and IgG-positive workers, albumin adducts were significantly higher versus workers without DA and those who were specific IgG negative. The odds ratio to be DA-positive was 57 times larger for workers with adduct levels above 230 fmol/mg. The odds ratio to be IgG-positive was 10 times larger for workers with adduct levels above 113 fmol/mg. Therefore, albumin adducts appear to be a good predictor of the biological effects. The albumin-adduct levels in workers without biological effects were in the range of the adduct levels found in previous studies of healthy MDI-factory and construction site workers.

  3. Chlorambucil-adducts in DNA analyzed at the oligonucleotide level using HPLC-ESI MS.

    PubMed

    Mohamed, Dalia; Mowaka, Shereen; Thomale, Jürgen; Linscheid, Michael W

    2009-08-01

    Chlorambucil (N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating drug belonging to the nitrogen mustard group and is widely used as an anticancer agent. As the antitumor activity of the nitrogen mustards is based on the formation of adducts with genomic DNA, calf thymus DNA-Chlorambucil adducts were the major target in this study. Calf thymus DNA was incubated with Chlorambucil to induce the formation of a wide variety of adducts. Subsequently, enzymatic digestion of the DNA was performed using Benzonase and Nuclease S1 aiming at the production of oligonucleotides. Separation and structure elucidation of the individual DNA-Chlorambucil adducts was achieved using HPLC interfaced to electrospray ionization ion trap mass spectrometry. Both trinucleotide and tetranucleotide Chlorambucil adducts were detected. The majority of the detected trinucleotide adducts involved monofunctional alkylation with guanine being the hotspot for alkylation. Only a few bifunctional trinucleotide adducts both intra- and interstrand cross-links were found. On the contrary, cross-linked adducts were the major detected tetranucleotides in which the intrastrand cross-links predominated over the interstrand cross-links. To a lesser extent, monofunctional guanine alkylated tetranucleotides were detected as well. With MS(n) experiments, the detailed structures of Chlorambucil adducts of the tri- and tetranucleotides were determined.

  4. Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

    PubMed Central

    Liu, Shuo; Wang, Yinsheng

    2016-01-01

    Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2–3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography-or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed. PMID:26204249

  5. Diallyl sulfide inhibits diethylstilbesterol-induced DNA adducts in the breast of female ACI rats.

    PubMed

    Green, M; Wilson, C; Newell, O; Sadrud-Din, S; Thomas, R

    2005-09-01

    Diethylstilbestrol (DES) is metabolized to reactive intermediates that produce DNA adducts and ultimately cancer. Diallyl sulfide (DAS) has been shown to inhibit the metabolism of several procarcinogens. The ability of DES to produce DNA adducts in microsomal, mitochondrial, and nuclear in vitro metabolic systems and in the breast of female ACI rats, as well as ability of DAS to inhibit DNA adducts were investigated. Microsomes, mitochondria, and nuclei isolated from breast tissue of female ACI rats were used to catalyze oxidation reactions. Female ACI rats were treated i.p. as follows: (1) corn oil, (2) 200mg/kg DES, (3) 200mg/kg DES/200mg/kg of DAS, (4) 200mg/kg DES/400mg/kg DAS. DES produced DNA adducts in each metabolic system. The relative adduct levels were 2.1 x 10(-4), 6.2 x 10(-6), and 2.9 x 10(-7) in microsomal, mitochondrial, and nuclear reactions, respectively. DAS inhibited DNA adducts in each metabolic system. The percent inhibition ranged from 86% in microsomes to 93% in nuclei. DES produced DNA adducts in mtDNA and nDNA. DAS completely inhibited the DES-induced mtDNA adducts and caused a dose dependent decrease in nDNA adduct formation. These findings suggest that DAS could inhibit DES-induced breast cancer by inhibiting its metabolism.

  6. Formation of DNA adducts from oil-derived products analyzed by 32P-HPLC.

    PubMed

    Akkineni, L K; Zeisig, M; Baranczewski, P; Ekström, L G; Möller, L

    2001-01-01

    The aim of this study was to investigate the genotoxic potential of DNA adducts and to compare DNA adduct levels and patterns in petroleum vacuum distillates, coal tar distillate, bitumen fume condensates, and related substances that have a wide range of boiling temperatures. An in vitro assay was used for DNA adduct analysis with human and rat S-9 liver extract metabolic activation followed by 32P-postlabeling and 32P-high-performance liquid chromatography (32p-HPLC). For petroleum distillates originating from one crude oil there was a correlation between in vitro DNA adduct formation and mutagenic index, which showed an increase with a distillation temperature of 250 degrees C and a peak around a distillation point of approximately 400 degrees C. At higher temperatures, the genotoxicity (DNA adducts and mutagenicity) rapidly declined to very low levels. Different petroleum products showed a more than 100-fold range in DNA adduct formation, with severely hydrotreated base oil and bitumen fume condensates being lowest. Coal tar distillates showed ten times higher levels of DNA adduct formation than the most potent petroleum distillate. A clustered DNA adduct pattern was seen over a wide distillation range after metabolic activation with liver extracts of rat or human origin. These clusters were eluted in a region where alkylated aromatic hydrocarbons could be expected. The DNA adduct patterns were similar for base oil and bitumen fume condensates, whereas coal tar distillates had a wider retention time range of the DNA adducts formed. Reference substances were tested in the same in vitro assay. Two- and three-ringed nonalkylated aromatics were rather low in genotoxicity, but some of the three- to four-ringed alkylated aromatics were very potent inducers of DNA adducts. Compounds with an amino functional group showed a 270-fold higher level of DNA adduct formation than the same structures with a nitro functional group. The most potent DNA adduct inducers of the 16

  7. Gas phase adduct reactions in MOCVD growth of GaN

    SciTech Connect

    Thon, A.; Kuech, T.F.

    1996-11-01

    Gas phase reactions between trimethylgallium (TMG) and ammonia were studied at high temperatures, characteristic to MOCVD of GaN reactors, by means of in situ mass spectroscopy in a flow tube reactor. It is shown, that a very fast adduct formation followed by elimination of methane occurs. The decomposition of TMG and the adduct-derived compounds are both first order and have similar apparent activation energy. The pre-exponential factor of the adduct decomposition is smaller, and hence is responsible for the higher full decomposition temperature of the adduct relative to that of TMG.

  8. Formation and characterization of carbon monoxide adducts of iron "twin coronet" porphyrins. Extremely low CO affinity and a strong negative polar effect on bound CO.

    PubMed

    Matsu-Ura, Mikiya; Tani, Fumito; Naruta, Yoshinori

    2002-03-06

    The carbon monoxide (CO) adducts of iron "twin coronet" porphyrins (TCPs) are characterized by UV-vis, resonance Raman (RR), IR, and 13C NMR spectroscopies. A superstructured porphyrin, designated as TCP, was used as a common framework for the four different types of iron complexes. TCP bears two binaphthalene bridges on each side and creates two hydrophobic pockets surrounded by the bulky aromatic rings. In the CO-binding cavities, the hydroxyl groups are oriented toward the center above the heme. The iron complexes investigated are as follows: TCP (which is without a covalently linked axial ligand), TCP-PY (which has a linked pyridine ligand), and TCP-TB and TCP-TG (both of which have a linked thiolate ligand). These complexes were synthesized as ferric forms and identified by the various spectroscopic methods. The UV-vis spectra of TCP-CO and TCP-PY-CO exhibit lambda(max) at 432, 546 and 428, 541 nm, respectively. On the other hand, the CO adducts of TCP-TB and TCP-TG show typical hyperporphyrin spectra for a thiolate-ligated iron(II) porphyrin-CO complex. In the RR spectra, the nu(Fe-CO) bands were observed at 506, 489 cm(-1) (TCP), 465 cm(-1) (TCP-PY), 458, 437 cm(-1) (TCP-TG) and 429 cm(-1) (TCP-TB). Compared with the reported nu(Fe-CO) frequencies of hemoproteins and their model systems, these observed values are unusually low. Further, abnormally high nu(C-O) bands are observed at 1990 cm(-1) (TCP-CO) and 2008 cm(-1) (TCP-PY-CO) in IR spectra. The lower nu(Fe-CO) and the higher nu(C-O) frequencies can be ascribed to the strong negative polar effect caused by the vicinal hydroxyl groups in the cavity. This prediction is further supported by the observation of significant 13C shieldings exhibited by TCP-CO (delta = 202.6 ppm) and TCP-PY-CO (delta = 202.3 ppm), in comparison to hemoproteins and other heme models. The CO affinity of TCP-PY (P1/2CO = 0.017 Torr at 25 C) is unusually lower than other heme models. The unique behavior of these CO adducts is

  9. Definition of bulky disease in early stage Hodgkin lymphoma in computed tomography era: prognostic significance of measurements in the coronal and transverse planes.

    PubMed

    Kumar, Anita; Burger, Irene A; Zhang, Zhigang; Drill, Esther N; Migliacci, Jocelyn C; Ng, Andrea; LaCasce, Ann; Wall, Darci; Witzig, Thomas E; Ristow, Kay; Yahalom, Joachim; Moskowitz, Craig H; Zelenetz, Andrew D

    2016-10-01

    Disease bulk is an important prognostic factor in early stage Hodgkin lymphoma, but its definition is unclear in the computed tomography era. This retrospective analysis investigated the prognostic significance of bulky disease measured in transverse and coronal planes on computed tomography imaging. Early stage Hodgkin lymphoma patients (n=185) treated with chemotherapy with or without radiotherapy from 2000-2010 were included. The longest diameter of the largest lymph node mass was measured in transverse and coronal axes on pre-treatment imaging. The optimal cut off for disease bulk was maximal diameter greater than 7 cm measured in either the transverse or coronal plane. Thirty patients with maximal transverse diameter of 7 cm or under were found to have bulk in coronal axis. The 4-year overall survival was 96.5% (CI: 93.3%, 100%) and 4-year relapse-free survival was 86.8% (CI: 81.9%, 92.1%) for all patients. Relapse-free survival at four years for bulky patients was 80.5% (CI: 73%, 88.9%) compared to 94.4% (CI: 89.1%, 100%) for non-bulky; Cox HR 4.21 (CI: 1.43, 12.38) (P=0.004). In bulky patients, relapse-free survival was not impacted in patients treated with chemoradiotherapy; however, it was significantly lower in patients treated with chemotherapy alone. In an independent validation cohort of 38 patients treated with chemotherapy alone, patients with bulky disease had an inferior relapse-free survival [at 4 years, 71.1% (CI: 52.1%, 97%) vs 94.1% (CI: 83.6%, 100%), Cox HR 5.27 (CI: 0.62, 45.16); P=0.09]. Presence of bulky disease on multidimensional computed tomography imaging is a significant prognostic factor in early stage Hodgkin lymphoma. Coronal reformations may be included for routine Hodgkin lymphoma staging evaluation. In future, our definition of disease bulk may be useful in identifying patients who are most appropriate for chemotherapy alone.

  10. In vitro screening of 50 highly prescribed drugs for thiol adduct formation--comparison of potential for drug-induced toxicity and extent of adduct formation.

    PubMed

    Gan, Jinping; Ruan, Qian; He, Bing; Zhu, Mingshe; Shyu, Wen C; Humphreys, W Griffith

    2009-04-01

    Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

  11. Sodiation-based in-source collision for profiling of pyranocoumarins in Radix Peucedani (Qianhu): utility of sodium adducts' stability with in-source collision.

    PubMed

    Yang, W; Li, Y; Kang, C; Zhao, H; Xiang, L; Li, C; Wang, Q

    2017-03-01

    Full scan mode of liquid chromatography-mass spectrometry equipped with an electrospray ionization source offers a chance on global detection of complicated components; however, the scan mode carries significant challenges in rapidly capturing information of analysts. Sodiation-based in-source collision was proposed here, as a technique for rapid detecting untargeted analytes in full scan analysis, which was based on the stability of sodium adducts and the nonselectivity of in-source collision. Then the technique was applied to profile of angular-type pyranocoumarins (APs) in Radix Peucedani, with full scan analysis performed at two specific in-source collision energy: a high energy 50 V that is tolerated by the sodium adducts of APs, and a low energy 10 V, at which abundant adducts were offered. The spectra list of two average mass spectra was exported, and stable ions were selected based on the intensity ratio of standards at the two collision energy. Then 27 plausible [M + Na](+) m/z values of APs were acquired after filtering the fragment ion and isotope ions and validating with [M + NH4 ](+) . Eighty-two APs finally were tentatively identified based on their accurate spectral data of MS(n) , fragmentation rules, and elution order regardless of their absolute configuration, which included 25 reported APs from Peucedanum praeruptorum Dunn. The technique provided a novel application of sodium adduct in qualitative analysis. And it was valuable for rapidly capturing information of analytes in full scan analysis, not only for APs but also for other compounds that could form sodium adducts. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Immunochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes.

    PubMed Central

    Uchida, K; Szweda, L I; Chae, H Z; Stadtman, E R

    1993-01-01

    We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8378358

  13. Formation and persistence of arylamine DNA adducts in vivo.

    PubMed Central

    Beland, F A; Kadlubar, F F

    1985-01-01

    Aromatic amines are urinary bladder carcinogens in man and induce tumors at a number of sites in experimental animals including the liver, mammary gland, intestine, and bladder. In this review, the particular pathways involved in the metabolic activation of aromatic amines are considered as well as the specific DNA adducts formed in target and nontarget tissue. Particular emphasis is placed on the following compounds: 1-naphthylamine, 2-naphthylamine, 4-aminobiphenyl, 4-acetylaminobiphenyl, 4-acetylamino-4'-fluorobiphenyl, 3,2'-dimethyl-4-aminobiphenyl, 2-acetylaminofluorene, benzidine, N-methyl-4-aminoazobenzene, 4-aminoazobenzene, and 2-acetylaminophenanthrene. PMID:4085422

  14. Adducts of rare-earth pivaloyltrifluoroacetonates with macrocyclic polyethers

    SciTech Connect

    Martynova, T.N.; Korchkov, V.P.; Nikulina, L.D.

    1986-07-01

    Adducts of lanthanide tris(pivaloyltrifluoroacetonates) with crown ethers having the formulas Ln(PTA)/sub 3/ x 18-crown-6 (Ln = La, Nd, Tb, Er, Lu) and Ln(PTA)/sub 3/ x dibenzo-18-crown-6 (Ln = Nd, Tb, Er) have been synthesized. The compounds obtained have been studied by the methods of elemental analysis, UV and IR spectroscopy, PMR, and mass spectroscopy. On the basis of the physicochemical properties and the spectra studied it has been concluded that the lanthanide tris(..beta..-diketonates) interact with the crown ethers.

  15. Acute adduction deficit in a 7-week-old infant.

    PubMed

    Jain, Sunila; Goulstine, David; Gottlob, Irene

    2002-12-01

    A 7-week-old infant with sudden onset adduction deficit and proptosis is reported. The main differential diagnoses included orbital myositis, orbital cellulitis, capillary haemangioma and rhabdomyosarcoma. A CT scan revealed a postseptal cellulitis-like picture with thickening of the medial rectus muscle. He was given a course of antibiotics, withholding steroids and biopsy. His condition resolved completely on high-dose antibiotics alone. To our knowledge this is the youngest patient with infectious orbital myositis and postseptal cellulitis described in the literature. The clinical course emphasizes the importance of administering sufficiently high doses of antibiotics.

  16. Tyrosine-lipid peroxide adducts from radical termination: para coupling and intramolecular Diels-Alder cyclization.

    PubMed

    Shchepin, Roman; Möller, Matias N; Kim, Hye-young H; Hatch, Duane M; Bartesaghi, Silvina; Kalyanaraman, Balaraman; Radi, Rafael; Porter, Ned A

    2010-12-15

    Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.

  17. Depurinating acylfulvene-DNA adducts: characterizing cellular chemical reactions of a selective antitumor agent.

    PubMed

    Gong, Jiachang; Vaidyanathan, V G; Yu, Xiang; Kensler, Thomas W; Peterson, Lisa A; Sturla, Shana J

    2007-02-21

    Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF-DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed.

  18. Formation and persistence of benzo(a)pyrene metabolite-DNA adducts.

    PubMed Central

    Stowers, S J; Anderson, M W

    1985-01-01

    Benzo(a)pyrene (BP) and other polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and are suspected to be carcinogenic in man. The in vivo formation of BP metabolite-DNA adducts has been characterized in a variety of target and nontarget tissues of mice and rabbits. Tissues included were lung, liver, forestomach, colon, kidney, muscle, and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct, and an unidentified adduct were also observed. The adduct levels are unexpectedly similar in all the tissues examined from the same BP-treated animal. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approximately 50% of those in lung and liver at each oral BP dose used. We have also examined adduct levels formed in vivo in several cell types of lung and liver. Macrophages, type II cells, and Clara cells from lung and hepatocytes and nonpparenchymal cells from liver were isolated from BP-treated rabbits. BPDEI-deoxyguanosine adduct was observed in each cell type and, moreover, the levels were similar in various cell types. These and previous results strongly suggest that DNA in many human tissues is continuously damaged from known exposure of humans to BP and other PAH. Moreover, DNA adducts formed from BP are persistent in lung and brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4085435

  19. Treatment of bulky stage IB and IIB cervical cancers with outpatient neutron brachytherapy, external pelvic radiation and extrafascial hysterectomy

    SciTech Connect

    Van Nagell, J.R.; Maruyama, Y.; Yoneda, J.; Donaldson, E.S.; Hanson, M.B.; Gallion, H.H.; Powell, D.E.; Kryscio, R.J.

    1986-01-01

    From January, 1977, to December, 1982, twenty-nine patients with bulky (>4 cms diameter) Stage IB or IIB cervical cancer were treated at the University of Kentucky Medical Center by a combination of out-patient neutron brachytherapy (Cf-252) and external pelvic radiation followed by extrafascial hysterectomy. Residual tumor was present in the hysterectomy specimens of 25 per cent. Complications during and following radiation therapy and surgery were minimal and included vaginal stenosis, proctitis, and hemorrhagic cystitis. The mean duration of hospitalization for surgery in these patients was 6.6 days (range 5-15 days) and postoperative morbidity was low. No patient required blood transfusion. Four patients developed urinary tract infections and two had superficial wound separations. Following treatment, patients were seen at monthly intervals for one year, every three months for two years, and every six months thereafter. No patient has been lost to follow-up. Two patients (7 per cent) developed tumor recurrence and have died of disease (1 of distant metastases; 1 local). The remaining 27 patients (93 per cent) are alive and well with no evidence of disease 24-89 months (mean 48 months) after therapy. No radiogenic fistulae or bowel obstruction were observed. These preliminary results suggest that the combination of outpatient neutron brachytherapy, external pelvic radiation, and extrafascial hysterectomy for patients with Stage IB and IIB cervical cancer is well tolerated. Complications associated with this treatment regimen have been minimal, and the recurrence rate is low. The duration of intracavitary neutron brachytherapy was short, and outpatient therapy was well received by patients.

  20. Effect of induction chemotherapy on estimated risk of radiation pneumonitis in bulky non–small cell lung cancer

    SciTech Connect

    Amin, Neha P.; Miften, Moyed; Thornton, Dale; Ryan, Nicole; Kavanagh, Brian; Gaspar, Laurie E

    2013-10-01

    Patients with bulky non–small cell lung cancer (NSCLC) may be at a high risk for radiation pneumonitis (RP) if treated with up-front concurrent chemoradiation. There is limited information about the effect of induction chemotherapy on the volume of normal lung subsequently irradiated. This study aims to estimate the reduction in risk of RP in patients with NSCLC after receiving induction chemotherapy. Between 2004 and 2009, 25 patients with Stage IV NSCLC were treated with chemotherapy alone (no surgery or radiation therapy [RT]) and had computed tomography (CT) scans before and after 2 cycles of chemotherapy. Simulated RT plans were created for the prechemotherapy and postchemotherapy scans so as to deliver 60 Gy to the thoracic disease in patients who had either a >20% volumetric increase or decrease in gross tumor volume (GTV) from chemotherapy. The prechemotherapy and postchemotherapy scans were analyzed to compare the percentage of lung volume receiving≥20 Gy (V20), mean lung dose (MLD), and normal tissue complication probability (NTCP). Eight patients (32%) had a GTV reduction >20%, 2 (8%) had GTV increase >20%, and 15 (60%) had stable GTV. In the 8 responders, there was an absolute median GTV decrease of 88.1 cc (7.3 to 351.6 cc) or a 48% (20% to 62%) relative reduction in tumor burden. One had >20% tumor progression during chemotherapy, yet had an improvement in dosimetric parameters postchemotherapy. Among these 9 patients, the median decrease in V20, MLD, and NTCP was 2.6% (p<0.01), 2.1 Gy (p<0.01), and 5.6% (p<0.01), respectively. Less than one-third of patients with NSCLC obtain >20% volumetric tumor reduction from chemotherapy alone. Even with that amount of volumetric reduction, the 5% reduced risk of RP was only modest and did not convert previously ineligible patients to safely receive definitive thoracic RT.

  1. Stage I-IIA Non-Bulky Hodgkin's Lymphoma. Is Further Distinction Based on Prognostic Factors Useful? The Stanford Experience

    SciTech Connect

    Advani, Ranjana H.; Hoppe, Richard T.; Maeda, Lauren S.; Baer, David M.; Mason, Joseph; Rosenberg, Saul A.; Horning, Sandra J.

    2011-12-01

    Purpose: In the United States, early-stage Hodgkin's lymphoma (HL) is defined as asymptomatic stage I/II non-bulky disease. European groups stratify patients to more intense treatment by considering additional unfavorable factors, such as age, number of nodal sites, sedimentation rate, extranodal disease, and elements of the international prognostic score for advanced HL. We sought to determine the prognostic significance of these factors in patients with early-stage disease treated at Stanford University Medical Center. Methods and Materials: This study was a retrospective analysis of 101 patients treated with abbreviated Stanford V chemotherapy (8 weeks) and 30-Gy (n = 84 patients) or 20-Gy (n = 17 patients) radiotherapy to involved sites. Outcomes were assessed after applying European risk factors. Results: At a median follow-up of 8.5 years, freedom from progression (FFP) and overall survival (OS) rates were 94% and 97%, respectively. From 33% to 60% of our patients were unfavorable per European criteria (i.e., German Hodgkin Study Group [GHSG], n = 55%; European Organization for Research and Treatment of Cancer, n = 33%; and Groupe d'Etudes des Lymphomes de l'Adulte, n = 61%). Differences in FFP rates between favorable and unfavorable patients were significant only for GHSG criteria (p = 0.02) with there were no differences in OS rates for any criteria. Five of 6 patients who relapsed were successfully salvaged. Conclusions: The majority of our patients deemed unfavorable had an excellent outcome despite undergoing a significantly abbreviated regimen. Application of factors used by the GHSG defined a less favorable subset for FFP but with no impact on OS. As therapy for early-stage disease moves to further reductions in therapy, these factors take on added importance in the interpretation of current trial results and design of future studies.

  2. Evaluation of time-dose and fractionation for sup 252 Cf neutrons in preoperative bulky/barrel-cervix carcinoma radiotherapy

    SciTech Connect

    Maruyama, Y.; Wierzbicki, J. )

    1990-12-01

    Time-dose fractionation factors (TDF) were calculated for 252Cf (Cf) neutron therapy versus 137Cs for intracavitary use in the preoperative treatment of bulky/barrel-shaped Stage IB cervix cancers. The endpoint assessed was gross and microscopic tumor eradication from the hysterectomy specimen. We reviewed the data obtained in clinical trials between 1976-1987 at the University of Kentucky Medical Center. Preoperative photon therapy was approximately 45 Gy of whole pelvis irradiation in 5 weeks for both 137Cs and Cf treated patients. 137Cs implant was done after pelvic irradiation x1 to a mean dose of 2104 +/- 36 cGy at point A at a dose rate of 50.5 cGy/h. There were 37.5% positive specimens. Using Cf intracavitary implants, dose varied from 109 to 459 neutron cGy in 1-2 sessions. Specimens were more frequently cleared of tumor (up to 100% at appropriate dose) and showed a dose-response relationship, both by nominal dose and by TDF adjusted analysis of dose, dose-rate, number of sessions, and overall time. Limited understanding of relative biological effectiveness, schedule, effect of implants, and dose rate all made it difficult to use TDF to study neutron effects. Relative biological effectiveness (RBE) was estimated and showed that for Cf, RBE was a complex function of treatment variables. In the pilot clinical studies, a value of 6.0 had been assumed. The present findings of RBE for tumor destruction are larger than those assumed. Cf was effective for cervix tumor therapy and produced control without significant side effects due to the brachytherapy method used. The TDF model was of limited value in the present analysis and more information is still needed for RBE, dose-rate, and fractionation effects for Cf neutrons to develop a more sophisticated and relevant model.

  3. Synthesis of gamma-hydroxybutenolides applying crossed aldol condensation in the presence of a bulky lewis acid and their anti-tumor activity.

    PubMed

    Yamano, Yumiko; Fujita, Yumi; Mizuguchi, Yukari; Nakagawa, Kimie; Okano, Toshio; Ito, Masayoshi; Wada, Akimori

    2007-09-01

    An improved synthesis of gamma-hydroxybutenolides 1a-d was achieved via crossed aldol condensation between aldehydes 2a-d and the protected gamma-hydroxy-beta-methylbutenolides 3 or 4 using the bulky Lewis acid, aluminum tris(2,6-diphenylphenoxide) (ATPH). Using this same methodology, the gamma-hydroxybutenolides 17a-d having various heteroaromatic rings were synthesized and their anti-tumor activities were evaluated.

  4. TANGO1 and Mia2/cTAGE5 (TALI) cooperate to export bulky pre-chylomicrons/VLDLs from the endoplasmic reticulum

    PubMed Central

    Santos, António J.M.; Nogueira, Cristina; Ortega-Bellido, Maria

    2016-01-01

    Procollagens, pre-chylomicrons, and pre–very low-density lipoproteins (pre-VLDLs) are too big to fit into conventional COPII-coated vesicles, so how are these bulky cargoes exported from the endoplasmic reticulum (ER)? We have shown that TANGO1 located at the ER exit site is necessary for procollagen export. We report a role for TANGO1 and TANGO1-like (TALI), a chimeric protein resulting from fusion of MIA2 and cTAGE5 gene products, in the export of pre-chylomicrons and pre-VLDLs from the ER. TANGO1 binds TALI, and both interact with apolipoprotein B (ApoB) and are necessary for the recruitment of ApoB-containing lipid particles to ER exit sites for their subsequent export. Although export of ApoB requires the function of both TANGO1 and TALI, the export of procollagen XII by the same cells requires only TANGO1. These findings reveal a general role for TANGO1 in the export of bulky cargoes from the ER and identify a specific requirement for TALI in assisting TANGO1 to export bulky lipid particles. PMID:27138255

  5. New ruthenium sensitizers featuring bulky ancillary ligands combined with a dual functioned coadsorbent for high efficiency dye-sensitized solar cells.

    PubMed

    Shi, Yongbo; Liang, Mao; Wang, Lina; Han, Hongyu; You, Lingshan; Sun, Zhe; Xue, Song

    2013-01-01

    Two ruthenium complexes featuring bulky ancillary ligands, XS48 and XS49, were synthesized and studied as dyes in dye-sensitized solar cells (DSCs). Both dyes exhibit higher solar-to-electrical energy conversion efficiency when compared to a commonly used N3 sensitizer under the same conditions. To examine the influence of the bulky ancillary ligands and alleviate the electron recombination in cells, we have developed a dual functioned truxene-based coadsorbent (MXD1) as an alternative candidate to chenodeoxycholic acid (CDCA). This coadsorbent not only effectively shields the back electron transfer from the TiO(2) to I(3)(-) ions but also enhances the light harvesting ability in the short wavelength regions. The photovoltaic performance of XS48-sensitized DSC was independent of the coadsorbents, while XS49 with large bulky ancillary ligand presented better performance when coadsorbent was employed. Interestingly, the simultaneous adsorption-to-sequential adsorption of XS48/49 and MXD1 has caused a notably improved photovoltage, which can be primarily ascribed to the enhanced dye adsorption and retardation of charge recombination. These results not only provide a new vision on how ancillary ligands affect the performance of ruthenium complexes but also open up a new way to achieve further efficiency enhancement of ruthenium complexes.

  6. TANGO1 and Mia2/cTAGE5 (TALI) cooperate to export bulky pre-chylomicrons/VLDLs from the endoplasmic reticulum.

    PubMed

    Santos, António J M; Nogueira, Cristina; Ortega-Bellido, Maria; Malhotra, Vivek

    2016-05-09

    Procollagens, pre-chylomicrons, and pre-very low-density lipoproteins (pre-VLDLs) are too big to fit into conventional COPII-coated vesicles, so how are these bulky cargoes exported from the endoplasmic reticulum (ER)? We have shown that TANGO1 located at the ER exit site is necessary for procollagen export. We report a role for TANGO1 and TANGO1-like (TALI), a chimeric protein resulting from fusion of MIA2 and cTAGE5 gene products, in the export of pre-chylomicrons and pre-VLDLs from the ER. TANGO1 binds TALI, and both interact with apolipoprotein B (ApoB) and are necessary for the recruitment of ApoB-containing lipid particles to ER exit sites for their subsequent export. Although export of ApoB requires the function of both TANGO1 and TALI, the export of procollagen XII by the same cells requires only TANGO1. These findings reveal a general role for TANGO1 in the export of bulky cargoes from the ER and identify a specific requirement for TALI in assisting TANGO1 to export bulky lipid particles.

  7. 32P-postlabeling analysis of adducts formed between DNA and safrole 2',3'-epoxide: absence of adduct formation in vivo.

    PubMed

    Qato, M K; Guenthner, T M

    1995-01-01

    We have used the 32P-postlabeling technique to examine the binding of safrole 2',3'-oxide to DNA. At least 8 covalent adducts are formed when calf thymus DNA is incubated with this oxygenated metabolite of safrole in vitro. However, no corresponding adducts are formed with liver DNA when whole animals are exposed to safrole 2',3'-oxide, or safrole itself. Although safrole 2',3'-oxide is readily formed in vivo, and is sufficiently reactive to covalently bind to DNA, it is probably not a factor in the in vivo genotoxicity of safrole. We also demonstrate that adducts with similar mobility to the major safrole 2',3'-oxide-DNA adduct are formed in vitro between safrole 2',3'-oxide and deoxyguanosine, and also between its chemical analogs allylbenzene 2',3'-oxide or estragole 2',3'-oxide and DNA.

  8. Turned head--adducted hip--truncal curvature syndrome.

    PubMed Central

    Hamanishi, C; Tanaka, S

    1994-01-01

    One hundred and eight neonates and infants who showed the clinical triad of a head turned to one side, adduction contracture of the hip joint on the occipital side of the turned head, and truncal curvature, which we named TAC syndrome, were studied. These cases included seven with congenital and five with late infantile dislocations of the hip joint and 14 who developed muscular torticollis. Forty one were among 7103 neonates examined by one of the authors. An epidemiological analysis confirmed the aetiology of the syndrome to be environmental. The side to which the head was turned and that of the adducted hip contracture showed a high correlation with the side of the maternal spine on which the fetus had been lying. TAC syndrome is an important asymmetrical deformity that should be kept in mind during neonatal examination, and may be aetiologically related to the unilateral dislocation of the hip joint, torticollis, and infantile scoliosis which develop after a vertex presentation. Images PMID:8048823

  9. Nonstoichiometric Adduct Approach for High-Efficiency Perovskite Solar Cells.

    PubMed

    Park, Nam-Gyu

    2017-01-03

    Since the groundbreaking report on a solid-state perovskite solar cell employing a methylammonium lead iodide-sensitized mesoporous TiO2 film and an organic hole conducting layer in 2012 by our group, the swift surge of perovskite photovoltaics opens a new paradigm in solar-cell research. As a result, ca. 1300 peer-reviewed research articles were published in 2015. In this Inorganic Chemistry Forum on Halide Perovskite, the researches with highlights of work on perovskite solar cells in my laboratory are reviewed. We have developed a size-controllable two-step spin-coating method and found that minimal nonradiative recombination in perovskite crystals could lead to high photovoltaic performance. A Lewis acid based adduct method and self-formed grain boundary process were developed for high-efficiency devices with reproducibility. A power conversion efficiency of 20.4% was achieved via grain boundary engineering based on a nonstoichiometric adduct approach. The incorporation of cesium in a formamidinium lead iodide perovskite was found to show better photostability and moisture-stability. A reduction in the dimensionality from a three-dimensitonal nanocrystal to a one-dimensional nanowire led to a hypsochromic shift of absorption and fluorescence. To enhance the charge-carrier transport and light-harvesting efficiency, a nanoarchitecture of oxide layers was proposed.

  10. Kinetics, mechanism and thermodynamics of bisulfite-aldehyde adduct formation

    SciTech Connect

    Olson, T.M.; Boyce, S.D.; Hoffmann, M.R.

    1986-04-01

    The kinetics and mechanism of bisulfite addition to benzaldehyde were studied at low pH in order to assess the importance of this reaction in stabilizing S(IV) in fog-, cloud-, and rainwater. Previously, the authors established that appreciable concentrations of the formaldehyde-bisulfite adduct (HMSA) are often present in fogwater. Measured HMSA concentrations in fogwater often do not fully account for observed excess S(IV) concentrations, however, so that other S(IV)-aldehyde adducts may be present. Reaction rates were determined by monitoring the disappearance of benzaldehyde by U.V. spectrophotometry under pseudo-first order conditions, (S(IV))/sub T/ >>(phi-CHO)/sub T/, in the pH range 0 - 4.4 at 25/sup 0/C. The equilibrium constant was determined by dissolving the sodium salt of the addition compound in a solution adjusted to pH 3.9, and measuring the absorbance of the equilibrated solution at 250 nm. A literature value of the extinction coefficient for benzaldehyde was used to calculate the concentration of free benzaldehyde. All solutions were prepared under an N/sub 2/ atmosphere using deoxygenated, deionized water and ionic strength was maintained at 1.0 M with sodium chloride.

  11. Nitropyrene: DNA binding and adduct formation in respiratory tissues.

    PubMed Central

    Jackson, M A; King, L C; Ball, L M; Ghayourmanesh, S; Jeffrey, A M; Lewtas, J

    1985-01-01

    Binding of 1-nitro (14C)pyrene (NP) or its metabolites to cellular DNA and protein in cultures of rabbit alveolar macrophages, lung tissue, and tracheal tissue was examined. DNA binding in tracheal tissue (136 +/- 18.3 pmole NP/mg DNA) was four to five times the levels measured in either lung tissue (38 +/- 9.4 pmole NP/mg DNA) or macrophages (26 +/- 7.5 pmole NP/mg DNA). Adduct analysis of DNA isolated from lung tissue incubated with 1-nitro[H3]pyrene in vitro resulted in the identification of 2 to 5% of the NP adducts as C8-deoxyguanosine 1-aminopyrene. NP was also bound to cellular protein in tracheal tissue and lung tissue, and at a lower level in macrophages. Cocultivation of the macrophages with lung and tracheal tissue decreased the DNA binding in tracheal tissue by 45%. Following intratracheal instillation of diesel particles (5 mg) vapor-coated with 14C-NP (380 ppm, 0.085 muCi/mg) particles into rats, 5-8% of the radioactivity remained in the lungs after 20 hr. Most of the diesel particles were also deposited in the lung. Examination of DNA and protein binding in this tissue showed 5 to 12% of the pulmonary 14C bound to protein and no detectable levels of 14C bound to DNA. PMID:3841313

  12. Inert Reassessment Document for Poly(oxyethylene) adducts of mixed phytosterols

    EPA Pesticide Factsheets

    Poly(oxyethy1ene) adducts of mixed phytosterols is uncategorized as to list classification status. Based upon the reasonable certainty of no harm safety finding, the List 4B classification for poly(oxyethy1ene) adducts of mixed phytosterols is affirmed.

  13. Condensed tannin-resorcinol adducts and their use in wood-laminating adhesives: An exploratory study

    Treesearch

    Richard W. Hemingway; R.E. Kreibich

    1984-01-01

    The reaction of a tannin extract (containing about 30% carbohydrate) from loblolly pine (Pinus taeda L.) bark (two parts) and resorcinol (one part) at 120°C for 24 h with acetic acid catalyst gave a product containing predominantly oligomeric procyanidin-4-resorcinol adducts (39%), unreacted resorcinol (22%), carbohydrate (20%). the resorcinol adduct...

  14. A hydrogen bond scaffold supported synthetic heme Fe(III)-O2(-) adduct.

    PubMed

    Mittra, Kaustuv; Chatterjee, Sudipta; Samanta, Subhra; Sengupta, Kushal; Bhattacharjee, Hridaynath; Dey, Abhishek

    2012-11-04

    A hydrogen bonded heme-Fe(III)-O(2)(-) adduct is stabilized and characterized using resonance Raman and EPR spectroscopy. The low O-O vibrations of this complex are quite different from those reported for other heme-Fe(III)-O(2)(-) adducts.

  15. Significance of DNA adduct studies in animal models for cancer molecular dosimetry and risk assessment.

    PubMed Central

    Beland, F A; Poirier, M C

    1993-01-01

    To elucidate the relationship between DNA adduct formation and tumorigenesis, a number of experiments have been conducted to measure DNA adducts in target tissues from experimental animals during continuous exposure to carcinogens. With aflatoxins, aromatic amines, and polycyclic aromatic hydrocarbons, tumor induction appears to be associated with the major DNA adduct detected, whereas with N-nitrosamines the response is normally correlated with minor forms of DNA damage. During continuous carcinogen administration, steady-state adduct concentrations are generally obtained in the target tissues, and there is often a linear correlation between the carcinogen concentration and the steady-state DNA adduct level. Exceptions exist when the mechanism of activation changes or with the onset of significant toxicity. Steady-state DNA adduct levels are often linearly related to the tumorigenic response. Carcinogen-induced cell proliferation occurs when significant deviations from linearity are observed. Because DNA adducts detected in humans are chemically identical to those found in experimental animals, DNA adduct data in animals may contribute to our understanding of human cancer risk. PMID:8319658

  16. 40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Toluene sulfonamide bis-phe-nol A... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

  17. 40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Toluene sulfonamide bis-phe-nol A... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

  18. 40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Toluene sulfonamide bis-phe-nol A... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

  19. 40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Toluene sulfonamide bis-phe-nol A... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

  20. Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap.

    PubMed

    Hardy, Micaël; Rockenbauer, Antal; Vásquez-Vivar, Jeannette; Felix, Christopher; Lopez, Marcos; Srinivasan, Satish; Avadhani, Narayan; Tordo, Paul; Kalyanaraman, B

    2007-07-01

    We report here the detection and characterization of spin adducts formed from the trapping of reactive oxygen species (superoxide and hydroxyl radicals) and glutathiyl and carbon-centered radicals by a newly synthesized nitrone, Mito-DEPMPO. This is a cationic nitrone spin trap with a triphenyl phosphonium cation conjugated to the DEPMPO analogue. The Mito-DEPMPO-OOH adduct, formed from the trapping of superoxide by Mito-DEPMPO, was enzymatically generated using xanthine/xanthine oxidase and neuronal nitric oxide synthase, and chemically generated by KO2 in 18-crown-6. The Mito-DEPMPO-OOH adduct exhibits an eight-line EPR spectrum with partial asymmetry arising from the alternate line-width effect. The half-life of the Mito-DEPMPO-OOH adduct is 2-2.5-times greater than that of the DEPMPO-OOH. The Mito-DEPMPO-SG adduct, formed from the trapping of glutathiyl radicals by Mito-DEPMPO, is 3-times more persistent than the analogue DEPMPO-SG adduct. In this study, we describe the EPR characterization of spin adducts formed from Mito-DEPMPO. The EPR parameters of Mito-DEPMPO adducts are distinctly different and highly characteristic. The detection of superoxide from an intact mitochondrion was feasible with Mito-DEPMPO but not with DEPMPO. We conclude that Mito-DEPMPO nitrone and its analogues are more effective than most nitrone spin traps for trapping superoxide, hydroxyl, and thiyl radicals formed in biological systems, including mitochondria.

  1. Depurinating naphthalene–DNA adducts in mouse skin related to cancer initiation

    PubMed Central

    Saeed, Muhammad; Higginbotham, Sheila; Gaikwad, Nilesh; Chakravarti, Dhrubajyoti; Rogan, Eleanor; Cavalieri, Ercole

    2015-01-01

    Naphthalene has been shown to be a weak carcinogen in rats. To investigate its mechanism of metabolic activation and cancer initiation, mice were topically treated with naphthalene or one of its metabolites, 1-naphthol, 1,2-dihydrodiolnaphthalene (1,2-DDN), 1,2-dihydroxynaphthalene (1,2-DHN), and 1,2-naphthoquinone (1,2-NQ). After 4 h, the mice were sacrificed, the treated skin was excised, and the depurinating and stable DNA adducts were analyzed. The depurinating adducts were identified and quantified by ultraperformance liquid chromatography/tandem mass spectrometry, whereas the stable adducts were quantified by 32P-postlabeling. For comparison, the stable adducts formed when a mixture of the four deoxyribonucleoside monophosphates was treated with 1,2-NQ or enzyme-activated naphthalene were also analyzed. The depurinating adducts 1,2-DHN-1-N3Ade and 1,2-DHN-1-N7Gua arise from reaction of 1,2-NQ with DNA. Similarly, the major stable adducts appear to derive from the 1,2-NQ. The depurinating DNA adducts are, in general, the most abundant. Therefore, naphthalene undergoes metabolic activation to the electrophilic ortho-quinone, 1,2-NQ, which reacts with DNA to form depurinating adducts. This is the same mechanism as other weak carcinogens, such as the natural and synthetic estrogens, and benzene. PMID:19619639

  2. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL..., ethylene oxide adduct. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  3. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL..., ethylene oxide adduct. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  4. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL..., ethylene oxide adduct. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  5. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL..., ethylene oxide adduct. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  6. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL..., ethylene oxide adduct. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  7. Immunohistochemical localization of trichloroacylated protein adducts in tetrachloroethene-treated mice.

    PubMed

    Green, S M; Khan, M F; Kaphalia, B S; Ansari, G A

    2001-05-25

    Tetrachloroethene (PCE), a common industrial solvent and environmental contaminant, is primarily used in the dry-cleaning industry. The toxicity of PCE has been linked to vision disorders, renal and hepatic cancer, and autoimmune diseases. Although the mechanism of toxicity is not fully understood, PCE forms trichloroacylated protein adducts in tissues where toxicity is known to occur. These adducts may be responsible for toxicity by altering the function of cellular proteins. Using Western blot analysis, formation of trichloroacylated protein adducts has been reported. To determine the localization of the adducts in a specific zone of a tissue, immunohistochemical staining was used in the study. An antiserum to trichloroacylated proteins was raised in rabbits and its specificity was established by enzyme-linked immunosorbent assay (ELISA). Female MRL-lpr/lpr and MRL +/+ mice were treated with PCE using a single 5-mmol/kg dose over 24 h or on every fourth day for 6 wk (total 20 doses). Formation of trichloroacylated protein adducts was observed in the liver, and localized to the centrilobular zones. Intensity and circumference of the staining around the central vein were much greater in subchronically treated mice than in acutely treated mice. No immunochemical reactivity was observed in any of the other tissues examined. This study shows that hepatic trichloroacylated protein adducts are localized in a region of the liver where PCE-mediated toxicity is known to occur. Immunohistochemical localization of these adducts and its association with PCE-induced toxicity support the contention that adducts may contribute to toxicity.

  8. Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions.

    PubMed

    Nikolantonaki, Maria; Jourdes, Michael; Shinoda, Kentaro; Teissedre, Pierre-Louis; Quideau, Stéphane; Darriet, Philippe

    2012-03-14

    HPLC-MS and (1)H, (13)C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.

  9. Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

    PubMed Central

    Luna, Leah G.; Green, Brett J.; Zhang, Fagen; Arnold, Scott M.; Siegel, Paul D.; Bartels, Michael J.

    2016-01-01

    4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results were obtained

  10. Chirped-Pulse and Cavity Based Fourier Transform Microwave Spectroscopy of the Methyl Lactate-Ammonia Adduct

    NASA Astrophysics Data System (ADS)

    Thomas, Javix; Sukhorukov, Oleksandr; Jaeger, Wolfgang; Xu, Yunjie

    2012-06-01

    The hydrogen bonded complex of ammonia with methyl lactate, a chiral alpha-hydroxyester, has been studied using rotational spectroscopy and high level ab initio calculations. Previous studies showed that methyl lactate can exist in a number of conformers. However, only the most stable one which has an intramolecular hydrogen bonded ring formed with its alcoholic hydroxyl and its carbonyl oxygen atom was detected experimentally An extensive ab initio search has been performed to locate all possible low energy conformers of the methyl lactate-ammonia contact pair. Five lowest energy conformers have been identified at the MP2/6-311++G(d,p) level. The lowest energy conformer favors an insertion arrangement, where ammonia is inserted into the existing intramolecular hydrogen bonded ring in the most stable methyl lactate conformer. Broadband scans for the rotational spectra of possible binary conformers have been carried out using a chirped-pulse Fourier transform microwave (FTMW) instrument. The most stable binary adduct was identified and assigned. The final frequency measurements have been done with a cavity based FTMW instrument. The spectrum observed shows complicated fine and hyperfine splitting patterns, likely due to the internal rotations of the methyl groups of methyl lactate and that of ammonia, as well as the 14N quadrupolar nucleus. The binary adduct with 15NH3 has also been studied to simplify the splitting pattern and to aid the assignments of the extensive splittings. The isotopic data and the fine and hyperfine structures will be discussed in terms of internal rotation dynamics and geometry of the hydrogen bonded adduct.

  11. DNA adducts in marine mussel and fresh water fishes living in polluted and unpolluted environments

    SciTech Connect

    Kurelec, B.; Checko, M.; Krca, S.; Garg, A.; Gupta, R.C. Baylor College of Medicine, Houston, TX )

    1988-09-01

    {sup 32}P-postlabeling analysis of DNA adducts in the digestive gland of marine mussel Mytilus galloprovincialis from polluted and unpolluted sites near Rovinj, Northern Adriatic, revealed that majority of adducts are caused by natural environmental factors rather than by man-made chemicals. The only pollutant-specific adducts were observed in a mussel exposed to seawater experimentally polluted with aminofluorene, and in a population of mussel living at a site heavily polluted with a waste waters of an oil refinery. Fresh water fish species Leuciscus cephalus, Barbus barbus, Abramis brama and Rutilus pigus virgo living in a polluted Sava River, Yugoslavia, or in its unpolluted tributary Korana River, have induced in their livers qualitatively identical and quantitatively similar DNA adducts. These DNA adducts had a species-specific patterns and their appearance was seasonally-dependent.

  12. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts.

    PubMed

    Aparicio, Tomas; Baer, Richard; Gottesman, Max; Gautier, Jean

    2016-02-15

    Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein-DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)-DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2-DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.

  13. Drug-Protein Adducts: Chemistry, Mechanisms of Toxicity, and Methods of Characterization.

    PubMed

    Gan, Jinping; Zhang, Haiying; Humphreys, W Griffith

    2016-12-19

    The formation of drug-protein adducts is considered an important feature in the pharmacological and toxicological profiles of many drugs. Mechanistic insights into the role of specific protein adduct formation in pharmacology and toxicology remain scarce, partly due to the availability of tools to identify and characterize the specific protein adducts, and partly due to the scarcity of relevant in vitro and in vivo predictive models. This review serves to provide a review on the current state of science on the chemistry, toxicology, and methods of detection and characterization of drug-protein adducts and to offer some perspective on the future directions of research into the role of protein adducts in drug effects and toxicity.

  14. Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa.

    PubMed

    Musser, S M; Pan, S S; Callery, P S

    1989-07-14

    High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated.

  15. Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes

    SciTech Connect

    Suh, Myungkoo

    1995-12-06

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7β, 8α-dihydoxy-9α, l0α-epoxy-7,8,9, 10-tetrahydrobenzo[α]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( ~25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant π-π stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G2 or G3 (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N2-dG in DNA isolated from the skin of mice treated topically with benzo[α]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG.

  16. White blood cell DNA adducts in a cohort of asthmatic children exposed to environmental tobacco smoke.

    PubMed

    Wilson, Stephen E; Talaska, Glenn; Kahn, Robert S; Schumann, Brenda; Khoury, Jane; Leonard, Anthony C; Lanphear, Bruce P

    2011-01-01

    Exposure to environmental tobacco smoke (ETS) leads to molecular damage in the form of DNA adducts. While lung cancer risk is higher among African Americans compared to White Americans, a few studies have tested for racial differences in DNA adducts among children exposed to ETS. The purpose of this study was to test whether African American children have higher DNA adducts levels compared to White children adjusted for ETS exposure. Data and biologic specimens were drawn from an existing cohort of 212 asthmatic children. These subjects participated in a 12-month ETS-reduction trial that employed HEPA air cleaners with active filter cartridges and sham filter cartridges. White blood cell (WBC) DNA was analyzed for DNA adducts using (32)P-postlabeling. We assessed ETS exposure using a validated air nicotine dosimeter. We determined the independent relationship between African American race and DNA adduct levels adjusted for ETS exposure and air cleaner use. The mean age of the subjects was 8.4 years; 55% were African American. There was no difference in DNA adduct levels between African American and White children (11.8 vs. 11.2 adducts per 10(9) nucleotides, p = 0.86), despite slightly higher levels of air nicotine exposure (3.4 vs. 2.2 μg/m(3), p = 0.14). African American children used their air cleaners less often than White children. We found that the best predictor of DNA adduct levels was the duration of air cleaner use (r = -0.133, p = 0.056). This association was independent of cartridge type. We did not see differences in adduct levels by race even after accounting for the level of ETS exposure. However, there was a marginal inverse association between air cleaner use and adducts. Additional research is required to understand this phenomenon.

  17. DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles.

    PubMed

    Ross, Jeffrey A; Nelson, Garret B; Mutlu, Esra; Warren, Sarah H; Gilmour, M Ian; DeMarini, David M

    2015-01-01

    Biodiesel and biodiesel-blend fuels offer a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel-exhaust particles (DEP) from petroleum diesel and soy biodiesel and correlated DNA adduct levels and mutagenicity in Salmonella TA100. We examined two different DEP from petroleum diesel (C-DEP and B0), one from soy bean oil biodiesel (B100) and one from combustion of a blend of 20% B100 and 80% B0 (B20) for in vitro DNA adduct-forming potential under oxidative or nitroreductive conditions in the presence of calf thymus DNA as well as in vivo in Salmonella TA100. The modified DNA was hydrolyzed and analyzed by (32)P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with PAH and nitro-PAH adducts. The types and quantities of DNA adducts produced by the two independent petroleum diesel DEP were similar, with both polycyclic aromatic hydrocarbon (PAH)- and nitro-PAH-derived adducts formed. Relative potencies for S9-mediated DNA adduct formation, either per mass of particulate or per MJ(th) energy consumed were B100 > B0 > B20. Soy biodiesel emissions induced DNA damage in the form of presumptive PAH and nitro-PAH DNA adducts that correlated with mutagenicity in Salmonella. B20 is the soy biodiesel used most commonly in the US, and it produced the lowest DNA adduct-emission factor, ∼50% that of petroleum diesel.

  18. White blood cell DNA adducts in a cohort of asthmatic children exposed to environmental tobacco smoke

    PubMed Central

    Talaska, Glenn; Kahn, Robert S.; Schumann, Brenda; Khoury, Jane; Leonard, Anthony C.; Lanphear, Bruce P.

    2010-01-01

    Purpose Exposure to environmental tobacco smoke (ETS) leads to molecular damage in the form of DNA adducts. While lung cancer risk is higher among African Americans compared to White Americans, a few studies have tested for racial differences in DNA adducts among children exposed to ETS. The purpose of this study was to test whether African American children have higher DNA adducts levels compared to White children adjusted for ETS exposure. Methods Data and biologic specimens were drawn from an existing cohort of 212 asthmatic children. These subjects participated in a 12-month ETS-reduction trial that employed HEPA air cleaners with active filter cartridges and sham filter cartridges. White blood cell (WBC) DNA was analyzed for DNA adducts using 32P-postlabeling. We assessed ETS exposure using a validated air nicotine dosimeter. We determined the independent relationship between African American race and DNA adduct levels adjusted for ETS exposure and air cleaner use. Results The mean age of the subjects was 8.4 years; 55% were African American. There was no difference in DNA adduct levels between African American and White children (11.8 vs. 11.2 adducts per 109 nucleotides, p = 0.86), despite slightly higher levels of air nicotine exposure (3.4 vs. 2.2 μg/m3, p = 0.14). African American children used their air cleaners less often than White children. We found that the best predictor of DNA adduct levels was the duration of air cleaner use (r = −0.133, p = 0.056). This association was independent of cartridge type. Conclusions We did not see differences in adduct levels by race even after accounting for the level of ETS exposure. However, there was a marginal inverse association between air cleaner use and adducts. Additional research is required to understand this phenomenon. PMID:20336464

  19. Accurate characterization of carcinogenic DNA adducts using MALDI tandem time-of-flight mass spectrometry

    NASA Astrophysics Data System (ADS)

    Barnes, Charles A.; Chiu, Norman H. L.

    2009-01-01

    Many chemical carcinogens and their in vivo activated metabolites react readily with genomic DNA, and form covalently bound carcinogen-DNA adducts. Clinically, carcinogen-DNA adducts have been linked to various cancer diseases. Among the current methods for DNA adduct analysis, mass spectroscopic method allows the direct measurement of unlabeled DNA adducts. The goal of this study is to explore the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to determine the identity of carcinogen-DNA adducts. Two of the known carcinogenic DNA adducts, namely N-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (dG-C8-PhIP) and N-(2'-deoxyguanosin-8yl)-4-aminobiphenyl (dG-C8-ABP), were selected as our models. In MALDI-TOF MS measurements, the small matrix ion and its cluster ions did not interfere with the measurements of both selected dG adducts. To achieve a higher accuracy for the characterization of selected dG adducts, 1 keV collision energy in MALDI-TOF/TOF MS/MS was used to measure the adducts. In comparison to other MS/MS techniques with lower collision energies, more extensive precursor ion dissociations were observed. The detection of the corresponding fragment ions allowed the identities of guanine, PhIP or ABP, and the position of adduction to be confirmed. Some of the fragment ions of dG-C8-PhIP have not been reported by other MS/MS techniques.

  20. Analysis of serum PAH`s and PAH adducts by LC/MS

    SciTech Connect

    McClure, P.C.; Barr, J.R.; Maggio, V.L.

    1995-12-31

    Polycyclic aromatic hydrocarbons are an important class of chemical carcinogens. Benzo[a]pyrene is the most extensively studied and best understood carcinogenic PAH It is believed that Benzo[a]pyrene is metabolized in vitro to the diol epoxide, Benzo[a]pyrene-7,8-dihydrodiol-9, 10-epoxide which then can react with various nucleophilic centers on DNA. The major alkylation product appears to be the reaction of the Benzo[a]pyrene diol epoxide with the N{sup 2} position of guanine sites on DNA. Methods that can measure exposure and biological response to carcinogens such as PAH`s are needed. Human Blood can be separated into plasma, lymphocytes, and red blood cells. The plasma should contain native PAH`s which may yield some useful information about recent exposure. The red blood cells contain hemoglobin and adducts of PAH`s. Hemoglobin has an average lifetime of 120 days so quantification of hemoglobin adducts should give an average of a persons exposure over four months. Also, the electrophilic metabolites that react with hemoglobin to form adducts are the same metabolites that form DNA adducts which can lead to mutations and cancer. Lymphocytes contain DNA and therefore DNA adducts. DNA adducts can be repaired by a series of enzymes so quantification of these adducts will only yield information about recent or non-repairable adducts. DNA adduct formation is believed to be the first important step in chemical carcinogenesis so quantification of these adducts should yield some information on exposure and a great deal of important data on biological response and risk from specific PAH`s.

  1. DNA adducts as a measure of lung cancer risk in humans exposed to polycyclic aromatic hydrocarbons.

    PubMed Central

    Kriek, E; Van Schooten, F J; Hillebrand, M J; Van Leeuwen, F E; Den Engelse, L; De Looff, A J; Dijkmans, A P

    1993-01-01

    Workers in the coking, foundry, and aluminum industry can be exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and are at increased risk for lung cancer, as are cigarette smokers. In recent years several studies on workers in the foundry and coking industries have been reported. In these studies, white blood cell(WBC) DNA was used for analysis of PAH-DNA adducts. Theoretically, DNA adduct formation is a more relevant biological parameter for assessing exposure risk than PAH in the work atmosphere, or the amount of a metabolite in the urine, because adduct levels reflect that part of the dose that escapes detoxification and binds to DNA. We analyzed WBC DNA from coke-oven workers and from workers in an aluminum production plant and demonstrated the presence of PAH-DNA adducts. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their WBC compared with 27% of the controls (p < 0.05), measured with ELISA. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. In the aluminum workers, no PAH-DNA adducts were detected by ELISA, although the benzo[a]pyrene concentrations in the work atmosphere were comparable to those of the coke-oven workers. The more sensitive 32P-postlabeling assay showed the presence of PAH-DNA adducts in 91% of the aluminum workers. There was no correlation of WBC adduct levels with the concentration of PAH in the work atmosphere. Recently we showed that total PAH-DNA adduct levels in WBC from lung cancer patients were much higher than those generally found in healthy smokers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8319662

  2. Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the 32P-postlabelling assay.

    PubMed

    Segerbäck, D; Vodicka, P

    1993-12-01

    The 32P-postlabelling assay for analysis of DNA adducts of chemical carcinogens has been applied in a large number of experimental animal and human studies. Most human studies have dealt with occupational and environmental exposures to polycyclic aromatic hydrocarbons (PAHs). The postlabelling assay does not allow direct chemical identification, and most studies with this method have not been performed in a quantitative way. Very little is therefore known about the identity and absolute levels of adducts, which are important contributors to the process of risk identification and quantitation. In the present study it was, therefore, decided to test some parameters suspected to affect recoveries of adducts in the phosphorylation step of the assay. For this purpose 12 different PAHs were reacted individually and in a mixture with DNA in the presence of a rat liver S9 metabolizing system. Different concentrations of ATP, calcium chloride and polynucleotide kinase were tested using the nuclease P1 enhancement. We found that each factor contributed to adduct recovery and that optimal conditions could be defined. Diluting the modified DNA samples up to 1000 times had little influence on the recoveries of adducts. Comparing the nuclease P1 and the butanol extraction procedures for adduct purification showed that both methods gave similar patterns and levels of major adducts. The absolute recoveries in postlabelling, based on 3H-binding of radiolabelled compounds, were for most of the tested compounds relatively low. The fact that the nuclease P1 and the butanol extraction procedures gave similar recoveries points towards common factor(s) involved in the reduction of the recovered adduct levels. Based on the observed recoveries the conclusion can be drawn that when postlabelling related adducts in human samples the true total adduct levels can be considerably underestimated, even if optimal conditions are used.

  3. Development of an immunochemical detection method for atrazine-induced albumin adducts.

    PubMed

    Dooley, Greg P; Hanneman, William H; Carbone, David L; Legare, Marie E; Andersen, Melvin E; Tessari, John D

    2007-07-01

    Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is one of the most commonly used herbicides in the United States. Exposures in rodent models have led to a host of biological effects, most notably the suppression of luteinizing hormone surge. Previously, we have reported that diaminochlorotriazine (DACT), an atrazine metabolite, forms a covalent adduct with rat hemoglobin at Cys-125. In the present study, we investigated the formation of a similar covalent adduct at Cys-34 of rat and human albumins following atrazine exposure using MALDI-TOF/TOF MS and adduct-specific immunochemical detection. Using mass spectrometry, a covalent adduct with a mass of 110 Da was located on Cys-34 of albumin from rats exposed to 20, 50, 100, and 200 mg/kg atrazine as well as rat and human albumins exposed in vitro to 90 microg/mL DACT. On the basis of the formation of the adduct in vitro, the adduct structure is a dechlorinated diaminochlorotriazine. To further study this unique protein adduction, we collaborated with Strategic Biosolutions Inc. to generate a polyclonal antibody specific for the DACT adduct and report its use for immunochemical detection. We detected adduct formation in purified serum albumin samples from rats given 5, 10, 20, 50, 100, and 200 mg/kg atrazine as well as rat and human albumins exposed in vitro to 90 microg/mL DACT by using immunochemical analysis. No adducts were detected in control animals or in the in vitro controls using our immunochemical detection method. In summary, these data report the development of a novel immunochemical detection system that could provide a rapid screening methodology for the detection of atrazine in exposed human populations.

  4. CYCLOPENTA-FUSED POLYCYCLIC AROMATIC HYDROCARBONS IN STRAIN A/J MOUSE LUNG: DNA ADDUCTS, ONCOGENE MUTATIONS, & TUMORIGENESIS

    EPA Science Inventory

    Cyclopenta-fused Polycyclic Aromatic Hydrocarbons in Strain AJJ Mouse Lung: DNA Adducts, Oncogene Mutations, and Tumorigenesis.

    We have examined the relationships between DNA adducts, Ki-ras oncogene mutations, DNA adducts, and adenoma induction in the lungs of strain A/J...

  5. ON BENZO[A]PYRENE DERIVED DNA ADDUCTS FORMED IN LUNG TISSUE OF MICE

    EPA Science Inventory

    On Benzo [a] pyrene Derived DNA Adducts Formed in Lung Tissue of Mice
    The previously identified major DNA adducts of benzo[a]pyrene (BP) in vitro and in vivo are the stable and unstable adducts formed by reaction of the bay-region diol epoxide of BP (BPDE) and BP radical catio...

  6. CYCLOPENTA-FUSED POLYCYCLIC AROMATIC HYDROCARBONS IN STRAIN A/J MOUSE LUNG: DNA ADDUCTS, ONCOGENE MUTATIONS, & TUMORIGENESIS

    EPA Science Inventory

    Cyclopenta-fused Polycyclic Aromatic Hydrocarbons in Strain AJJ Mouse Lung: DNA Adducts, Oncogene Mutations, and Tumorigenesis.

    We have examined the relationships between DNA adducts, Ki-ras oncogene mutations, DNA adducts, and adenoma induction in the lungs of strain A/J...

  7. ON BENZO[A]PYRENE DERIVED DNA ADDUCTS FORMED IN LUNG TISSUE OF MICE

    EPA Science Inventory

    On Benzo [a] pyrene Derived DNA Adducts Formed in Lung Tissue of Mice
    The previously identified major DNA adducts of benzo[a]pyrene (BP) in vitro and in vivo are the stable and unstable adducts formed by reaction of the bay-region diol epoxide of BP (BPDE) and BP radical catio...

  8. The formation of covalent adducts between benzo(a)pyrenediol epoxide and RNA: Structural analysis by mass spectrometry

    SciTech Connect

    Yamamoto, J.; Subramaniam, R.; Wolfe, A.R.; Meehan, T. )

    1990-04-24

    Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention time with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribionucleotides were poly(G) > yeast RNA > poly(A) > poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Major fragmentation pathways resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions.

  9. DNA adducts in hematopoietic tissues and blood of the mummichog (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, Virginia.

    PubMed

    Rose, W L; French, B L; Reichert, W L; Faisal, M

    2000-01-01

    Hydrophobic DNA adducts were examined in liver, anterior kidney, spleen, and blood of tumor-prone mummichog (Fundulus heterclitus) from the creosote-contaminated Atlantic Wood (AW) site (Elizabeth River, Virginia). DNA adducts eluted in a diagonal radioactive zone, characteristic of polycyclic aromatic hydrocarbon exposure, in all examined tissues of AW fish. Mummichog demonstrated significantly higher levels of DNA adducts in spleen (394 +/- 109 nmol adducts/mol nucleotides) than in liver (201 +/- 77 nmol adducts/mol nucleotides) or anterior kidney (211 +/- 68 nmol adducts/mol nucleotides; P = 0.036). The levels of DNA adducts in the pooled blood (pool of four) were 142 nmol adducts/mol nucleotides. DNA adducts were not detected in the liver, anterior kidney, spleen and blood of fish collected from the reference site (< 2 nmol adducts/mol nucleotides). The high levels of DNA adducts detected in tissues of AW mummichog may be linked to the increased cancer incidence and immunosuppression in this population.

  10. DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats.

    PubMed

    Bieler, Christian A; Cornelius, Michael G; Klein, Reinhold; Arlt, Volker M; Wiessler, Manfred; Phillips, David H; Schmeiser, Heinz H

    2005-10-10

    3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the

  11. Hypervalent adducts of chalcogen-containing peri-substituted naphthalenes; reactions of sulfur, selenium, and tellurium with dihalogens.

    PubMed

    Knight, Fergus R; Fuller, Amy L; Bühl, Michael; Slawin, Alexandra M Z; Woollins, J Derek

    2010-08-16

    A range of structurally diverse compounds 1-15 {Nap[SPh](2).Br(4) (Nap = naphthalene-1,8-diyl); Nap[SePh][EPh].Br(4) (E = Se, S); Nap[SePh](2).I(2); Nap[SePh][EPh].3/2I(2) (E = Se, S); Nap[TePh][G].X(2) (G = SePh, SPh, Br, I; X = Br, I); and [Nap(PPh(2)OH)(SPh)](+)Br(3)(-)} formed from the reactions between peri-substituted naphthalene chalcogen donors D1-D8 {Nap[ER][E'R] (ER/E'R = SPh, SePh, TePh); Nap[TePh][X] (X = Br, I); and Nap[PPh(2)][SPh]} and dibromine and diiodine were characterized by X-ray crystallography and where possible by multinuclear NMR, IR, and MS. X-ray data for 1-15 were analyzed by naphthalene ring torsions, peri-atom displacement, splay angle magnitude, peri-distance, aromatic ring orientations, and quasi-linear three-body arrangements. The hypervalent linear moieties are considered in the context of the charge-transfer model and the 3c-4e model introduced by Pimentel and Rundle. In general, the conformation of the final products obeyed the rule based on charge-transfer that "seesaw" (X-ER(2)-X, 10-E-4) adducts arise when the halogen (X) is more electronegative than the chalcogen (E), and if the converse is true then, CT "spoke" (X-X-ER(2), 8-E-3) adducts are formed. Upon treatment with dibromine, selenium donor compounds D2 {Nap[SePh](2)} and D3 {Nap[SePh][SPh]} afford unusual tribromide salts of bromoselenyl cations containing a hypervalent X-E...E' 3c-4e type interaction. Upon treatment with diiodine, D2 and D3 form "Z-shaped", "extended spoke" adducts containing an uncommon 2:3 donor/chalcogen ratio and incorporating chains of I(2) held together by rare I...I interactions. As expected, "seesaw" 10-E-4 adducts are formed following the reaction of Te donors D4-D7 {Nap[TePh][X] (X = Br, I); Nap[TePh][EPh] (E = Se, S)} with the dihalogens. Naphthalene distortion in general is comparable between respective donor compounds and products 1-15. Ionic species 2 and 3 display a noticeable reduction in molecular distortion explained by the relief of

  12. Structures of exocyclic R,R- and S,S-N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine adducts induced by 1,2,3,4-diepoxybutane.

    PubMed

    Kowal, Ewa A; Seneviratne, Uthpala; Wickramaratne, Susith; Doherty, Kathleen E; Cao, Xiangkun; Tretyakova, Natalia; Stone, Michael P

    2014-05-19

    1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette smoke, and is classified as a human carcinogen. It is oxidized by cytochrome P450 to form 1,2,3,4-diepoxybutane (DEB); DEB bis-alkylates the N(6) position of adenine in DNA. Two enantiomers of bis-N(6)-dA adducts of DEB have been identified: R,R-N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (R,R-DHB-dA), and S,S-N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (S,S-DHB-dA) [ Seneviratne , U. , Antsypovich , S. , Dorr , D. Q. , Dissanayake , T. , Kotapati , S. , and Tretyakova , N. ( 2010 ) Chem. Res. Toxicol. 23 , 1556 -1567 ]. Herein, the R,R-DHB-dA and S,S-DHB-dA adducts have been incorporated into the 5'-d(C(1)G(2)G(3)A(4)C(5)X(6)A(7)G(8)A(9)A(10)G(11))-3':5'-d(C(12)T(13)T(14)C(15)T(16)T(17)G(18)T(19)C(20)C(21)G(22))-3' duplex [X(6) = R,R-DHB-dA (R(6)) or S,S-DHB-dA (S(6))]. The structures of the duplexes were determined by molecular dynamics calculations, which were restrained by experimental distances obtained from NMR data. Both the R,R- and S,S-DHB-dA adducts are positioned in the major groove of DNA. In both instances, the bulky 3,4-dihydroxypyrrolidine rings are accommodated by an out-of-plane rotation about the C6-N(6) bond of the bis-alkylated adenine. In both instances, the directionality of the dihydroxypyrrolidine ring is evidenced by the pattern of NOEs between the 3,4-dihydroxypyrrolidine protons and DNA. Also in both instances, the anti conformation of the glycosyl bond is maintained, which combined with the out-of-plane rotation about the C6-N(6) bond, allows the complementary thymine, T(17), to remain stacked within the duplex, and form one hydrogen bond with the modified base, between the imine nitrogen of the modified base and the T(17) N3H imino proton. The loss of the second Watson-Crick hydrogen bonding interaction at the lesion sites correlates with the lower thermal stabilities of the R,R- and S,S-DHB-dA duplexes, as

  13. Vitamin A-aldehyde adducts: AMD risk and targeted therapeutics

    PubMed Central

    Sparrow, Janet R.

    2016-01-01

    Although currently available treatment options for age-related macular degeneration (AMD) are limited, particularly for atrophic AMD, the identification of predisposing genetic variations has informed clinical studies addressing therapeutic options such as complement inhibitors and anti-inflammatory agents. To lower risk of early AMD, recommended lifestyle interventions such as the avoidance of smoking and the intake of low glycemic antioxidant-rich diets have largely followed from the identification of nongenetic modifiable factors. On the other hand, the challenge of understanding the complex relationship between aging and cumulative damage leading to AMD has fueled investigations of the visual cycle adducts that accumulate in retinal pigment epithelial (RPE) cells and are a hallmark of aging retina. These studies have revealed properties of these compounds that provide insights into processes that may compromise RPE and could contribute to disease mechanisms in AMD. This work has also led to the design of targeted therapeutics that are currently under investigation. PMID:27071115

  14. Rotational Spectra of Adducts of Formaldehyde with Freons

    NASA Astrophysics Data System (ADS)

    Qian, Gou; Feng, Gang; Evangelisti, Luca; Caminati, W.; Lopez, Montserrat Vallejo; Lesarri, Alberto; Cocinero, Emilio

    2013-06-01

    The rotational spectra of three 1:1 complexes of formaldehyde (H_{2}CO) with freons, i.e. difluoromethane (CH_{2}F_{2}), fluorochloromethane (CH_{2}FCl) and trifluorochloromethane (CF_{3}Cl), have been observed and assigned using pulsed jet Fourier transform microwave technique. Several isotopologues (including some ^{13}C species) have been measured in natural abundance. The tunnelling splittings have been measured in the first two adducts with relative intensity 1:3, due to the internal rotation of the formaldehyde moity along its symmetry axis. The barriers to this motion have been estimated by using a flexible model. For the latter two complexes, each of transition displays the hyperfine structures due to the quadrupolar effects of ^{35}Cl (^{37}Cl) nucleus. The dissociation energy has been estimated within the pseudo-diatomic approximation for all three complexes.

  15. Non Covalent Interactions and Internal Dynamics in Adducts of Freons

    NASA Astrophysics Data System (ADS)

    Caminati, Walther; Gou, Qian; Evangelisti, Luca; Feng, Gang; Spada, Lorenzo; Vallejo-López, Montserrat; Lesarri, Alberto; Cocinero, Emilio J.

    2014-06-01

    The complexation of chlorofluorocarbons (CFCs) with atmospheric water and pollutants of the atmosphere affects their reactivity and it seems to accelerate, for example, the decomposition rate of freons in the atmosphere [1]. For this reason we characterized shapes, stabilities, nature of the non-covalent interactions, structures and internal dynamics of a number of complexes of CFCs with water and of their dimers or oligomers by rotational spectroscopy. It has been found that hydrogenated CFCs form adducts with other molecules through weak hydrogen bonds (WHBs). Their C-H groups can act as proton donors, enhanced by the electron withdrawing of the halogen atoms, interacting with the electron rich regions of the partner molecules [2]. Also in adducts or oligomers of hydrogenated CFCs the monomer units are held together by nets of WHBs [3]. When CFCs are perhalogenated, the positive electrostatic region ("σ-hole") can interact electrostatically with negative sites of another, or of the same molecular entity, giving rise, according to IUPAC, to the so called halogen bond (HaB). However, it has been observed that when the perhalogenated CFCs has a Π electron system, a lone pair•••Π interaction (Bürgi-Dunitz) is favoured [4]. We describe here the HaBs that CF4 and CF3Cl form with a variety of partner molecules such as water, ammonia, dimethyl ether, etc. Important spectroscopic features outline strong dynamics effects taking place in this kind of complex. References [1] V. Vaida, H. G. Kjaergaard, K. J. Feierabend, Int. Rev. Phys. Chem. 22 (2003) 203. [2] See, for example: W. Caminati, S. Melandri, A. Maris, P. Ottaviani, Angew. Chem. Int. Ed. 45 (2006) 2438. [3] G. Feng, L. Evangelisti, I. Cacelli, L. Carbonaro, G. Prampolini, W. Caminati, Chem. Commun. 50 (2014) 171. [4] Q. Gou, G. Feng, L. Evangelisti, W. Caminati, Angew. Chem. Int. Ed. 52 (2013) 52 11888.

  16. Formation of monofunctional cisplatin-DNA adducts in carbonate buffer.

    PubMed

    Binter, Alexandra; Goodisman, Jerry; Dabrowiak, James C

    2006-07-01

    Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768-12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8mM HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 5mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8mM carbonate buffer, 5mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

  17. Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells.

    PubMed

    Martyniuk, Christopher J; Feswick, April; Fang, Bin; Koomen, John M; Barber, David S; Gavin, Terrence; Lopachin, Richard M

    2013-06-07

    Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. Protein adduct-trapping by hydrazinophthalazine drugs: mechanisms of cytoprotection against acrolein-mediated toxicity.

    PubMed

    Burcham, Philip C; Fontaine, Frank R; Kaminskas, Lisa M; Petersen, Dennis R; Pyke, Simon M

    2004-03-01

    Acrolein is a highly toxic aldehyde involved in a number of diseases as well as drug-induced toxicities. Its pronounced toxicity reflects the readiness with which it forms adducts in proteins and DNA. As a bifunctional electrophile, initial reactions between acrolein and protein generate adducts containing an electrophilic center that can participate in secondary deleterious reactions (e.g., cross-linking). We hypothesize that inactivation of these reactive protein adducts with nucleophilic drugs may counteract acrolein toxicity. Because we previously observed that 1-hydrazinophthalazine (hydralazine) strongly diminishes the toxicity of the acrolein precursor allyl alcohol, we explored the possibility that hydralazine targets reactive acrolein adducts in proteins. We report that hydralazine abolished the immunoreactivity of an acrolein-modified model protein (bovine serum albumin), but only if the drug was added to the protein within 30 min of commencing modification by acrolein. The ability of a range of carbonyl-trapping drugs to interfere with "early" events in protein modification strongly correlated with their protective potencies against allyl alcohol toxicity in hepatocytes. In mass spectrometry studies using a model lysine-containing peptide, hydralazine rapidly formed hydrazones with Michael adducts generated by acrolein. Using an antibody raised against such ternary drug-acrolein-protein complexes in Western blotting experiments, clear adduct-trapping was evident in acrolein-preloaded hepatocytes exposed to cytoprotective concentrations of hydralazine ranging from 2 to 50 microM. These novel findings begin to reveal the molecular mechanisms whereby hydralazine functions as an efficient "protein adduct-trapping" drug.

  19. Depurinating estrogen–DNA adducts in the etiology and prevention of breast and other human cancers

    PubMed Central

    Cavalieri, Ercole L; Rogan, Eleanor G

    2015-01-01

    Experiments on estrogen metabolism, formation of DNA adducts, mutagenicity, cell transformation and carcinogenicity have led to and supported the hypothesis that the reaction of specific estrogen metabolites, mostly the electrophilic catechol estrogen-3,4-quinones, with DNA can generate the critical mutations to initiate breast and other human cancers. Analysis of depurinating estrogen–DNA adducts in urine demonstrates that women at high risk of, or with breast cancer, have high levels of the adducts, indicating a critical role for adduct formation in breast cancer initiation. Men with prostate cancer or non-Hodgkin lymphoma also have high levels of estrogen–DNA adducts. This knowledge of the first step in cancer initiation suggests the use of specific antioxidants that can block formation of the adducts by chemical and biochemical mechanisms. Two antioxidants, N-acetylcysteine and resveratrol, are prime candidates to prevent breast and other human cancers because in various in vitro and in vivo experiments, they reduce the formation of estrogen–DNA adducts. PMID:20021210

  20. DNA adducts in carp exposed to artificial diesel-2 oil slicks.

    PubMed

    Kurelec, B; Garg, A; Krca, S; Britvić, S; Lucić, D; Gupta, R C

    1992-05-01

    In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by 32P-postlabeling in carp free-swimming in these tanks. 'Clusters' of lipophilic DNA adducts were detected, with five major and numerous minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to greater than 3000 amol/microgram DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (approximately 10,000 amol/microgram DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.

  1. Detection and characterization of human serum antibodies to polycyclic aromatic hydrocarbon diol-epoxide DNA adducts.

    PubMed Central

    Newman, M J; Light, B A; Weston, A; Tollurud, D; Clark, J L; Mann, D L; Blackmon, J P; Harris, C C

    1988-01-01

    The presence of serum antibodies to the diol-epoxide DNA adducts of representative polycyclic aromatic hydrocarbons (PAH), chrysene, benz[a]anthracene and benzo[a]pyrene, was determined by ELISA using serum samples obtained from normal healthy individuals. Antibodies that reacted against PAH adducted-DNA, but not against PAH-adducted protein, were found in the serum of approximately 40% of the test individuals. Specificity analysis of the antibodies demonstrated that serological cross-reactions between the benzo[a]pyrene and the chrysene diol-epoxide adducts were present. Similar cross-reactivity between the benz[a]anthracene and the chrysene adducts was observed. Sera containing antibodies that were apparently specific for each of the three PAH-DNA adducts were also identified. The presence of antibodies to PAH-DNA adducts indicates both past exposure to these carcinogenic PAH and their metabolic activation to the DNA damaging metabolites. These antibodies may prove to be useful in both retrospective and prospective epidemiological studies of various diseases associated with PAH exposure. PMID:3392204

  2. Conformational Properties of Equilenin-DNA Adducts: Stereoisomer and Base Effects

    PubMed Central

    Ding, Shuang; Shapiro, Robert; Cai, Yuqin; Geacintov, Nicholas E.; Broyde, Suse

    2008-01-01

    Equilin and equilenin, components of the hormone replacement therapy drug Premarin, can be metabolized to the catechol 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dC, dA and dG to form unusual stable cyclic adducts, which have been found in human breast tumor tissue. Four stereoisomeric adducts have been identified for each base. These twelve Premarin-derived adducts provide a unique opportunity for analyzing effects of stereochemistry and base damage on DNA structure, and consequently its function. Our computational studies have shown that these adducts, with obstructed Watson-Crick hydrogen bond edges and near-perpendicular ring systems, have limited conformational flexibility, and near-mirror image conformations in stereoisomer pairs. The dC and dA adducts can adopt major and minor groove positions in the double helix, but the dG adducts are positioned only in the major groove. In all cases, opposite orientations of the equilenin rings with respect to the 5'→3' direction of the damaged strand are found in stereoisomer pairs derived from the same base, and no Watson-Crick pairing is possible. However, detailed structural properties in DNA duplexes are distinct for each stereoisomer of each damaged base. These differences may underlie observed differential stereoisomer and base-dependent mutagenicities and repair susceptibilities of these adducts. PMID:18416538

  3. Polycyclic aromatic hydrocarbon-DNA adducts and the CYP1A1 restriction fragment length polymorphism

    SciTech Connect

    Shields, P.G.; Bowman, E.D.; Weston, A.; Harris, C.C.; Sugimura, H.; Caporaso, N.E.; Petruzzelli, S.F. ); Trump, B.F. )

    1992-11-01

    Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the [sup 32]P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a US lung cancer case-control study, no association with lung cancer was found. 17 refs., 3 figs.

  4. Effect of Foot Progression Angle and Lateral Wedge Insole on a Reduction in Knee Adduction Moment.

    PubMed

    Tokunaga, Ken; Nakai, Yuki; Matsumoto, Ryo; Kiyama, Ryoji; Kawada, Masayuki; Ohwatashi, Akihiko; Fukudome, Kiyohiro; Ohshige, Tadasu; Maeda, Tetsuo

    2016-10-01

    This study evaluated the effect of foot progression angle on the reduction in knee adduction moment caused by a lateral wedged insole during walking. Twenty healthy, young volunteers walked 10 m at their comfortable velocity wearing a lateral wedged insole or control flat insole in 3 foot progression angle conditions: natural, toe-out, and toe-in. A 3-dimensional rigid link model was used to calculate the external knee adduction moment, the moment arm of ground reaction force to knee joint center, and the reduction ratio of knee adduction moment and moment arm. The result indicated that the toe-out condition and lateral wedged insole decreased the knee adduction moment in the whole stance phase. The reduction ratio of the knee adduction moment and the moment arm exhibited a close relationship. Lateral wedged insoles decreased the knee adduction moment in various foot progression angle conditions due to decrease of the moment arm of the ground reaction force. Moreover, the knee adduction moment during the toe-out gait with lateral wedged insole was the smallest due to the synergistic effect of the lateral wedged insole and foot progression angle. Lateral wedged insoles may be a valid intervention for patients with knee osteoarthritis regardless of the foot progression angle.

  5. Effect of exercise and gait retraining on knee adduction moment in people with knee osteoarthritis.

    PubMed

    Khalaj, Nafiseh; Abu Osman, Noor A; Mokhtar, Abdul H; Mehdikhani, Mahboobeh; Wan Abas, Wan A B

    2014-02-01

    The knee adduction moment represents the medial knee joint load, and greater value is associated with higher load. In people with knee osteoarthritis, it is important to apply proper treatment with the least side effects to reduce knee adduction moment and, consequently, reduce medial knee joint load. This reduction may slow the progression of knee osteoarthritis. The research team performed a literature search of electronic databases. The search keywords were as follows: knee osteoarthritis, knee adduction moment, exercise program, exercise therapy, gait retraining, gait modification and knee joint loading. In total, 12 studies were selected, according to the selection criteria. Findings from previous studies illustrated that exercise and gait retraining programs could alter knee adduction moment in people with knee osteoarthritis. These treatments are noninvasive and nonpharmacological which so far have no or few side effects, as well as being low cost. The results of this review revealed that gait retraining programs were helpful in reducing the knee adduction moment. In contrast, not all the exercise programs were beneficial in reducing knee adduction moment. Future studies are needed to indicate best clinical exercise and gait retraining programs, which are most effective in reducing knee adduction moment in people with knee osteoarthritis.

  6. Malondialdehyde/Acetaldehyde Adduct (MAA) is the Dominant Epitope Following MDA Modification of Proteins in Atherosclerosis

    PubMed Central

    Duryee, Michael J.; Klassen, Lynell W.; Schaffert, Courtney S.; Tuma, Dean J.; Hunter, Carlos D.; Garvin, Robert P.; Anderson, Daniel R.; Thiele, Geoffrey M.

    2010-01-01

    Antibodies to malondialdehyde (MDA) modified macromolecules (adducts) have been detected in the serum of patients with atherosclerosis and correlate with the progression of this disease. However, the epitope and its formation have not been characterized. Studies have shown that excess MDA can be degraded to acetaldehyde which combines with proteins to from a stable dihydropyridine adduct. To investigate, mice were immunized with (MDA) adducts in the absence of adjuvant and showed an increase in antibodies to MDA adducts and the carrier protein as the concentration of MDA was increased. In fact, a number of the commercially available antibodies to MDA modified proteins were able to be inhibited by a chemical analogue hexyl-MAA. Also, MDA/MAA adducts were detected in the serum and aortic tissue of JCR diabetic/atherosclerotic rats. These studies determined that commercially available antibodies to MDA were shown to predominantly react with the MAA adduct and are present in the JCR model of atherosclerosis in both the serum and aortic tissue. Therefore, the immune response to MDA modified proteins is most likely to the dihydropyridine structure (predominant epitope in MAA), and suggests that MAA adducts may be playing a role in the development and/or progression of atherosclerosis. PMID:20696236

  7. PROTEIN TARGETS OF ACRYLAMIDE ADDUCT FORMATION IN CULTURED RAT DOPAMINERGIC CELLS

    PubMed Central

    Martyniuk, Christopher J.; Feswick, April; Fang, Bin; Koomen, John M.; Barber, David S.; Gavin, Terrence; LoPachin, Richard M.

    2013-01-01

    Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism. PMID:23566896

  8. Formation of DNA adducts in the skin of psoriasis patients, in human skin in organ culture, and in mouse skin and lung following topical application of coal-tar and juniper tar.

    PubMed

    Schoket, B; Horkay, I; Kósa, A; Páldeák, L; Hewer, A; Grover, P L; Phillips, D H

    1990-02-01

    Preparations of coal-tar and juniper tar (cade oil) that are used in the treatment of psoriasis are known to contain numerous potentially carcinogenic polycyclic aromatic hydrocarbons (PAH). Evidence of covalent binding to DNA by components of these mixtures was sought in a) human skin biopsy samples from 12 psoriasis patients receiving therapy with these agents, b) human skin explants maintained in organ culture and treated topically with the tars, and c) the skin and lungs of mice treated with repeated doses of the formulations following the regimen used in the clinic. DNA was isolated from the human and mouse tissues and digested enzymically to mononucleotides. 32P-Post-labeling analysis revealed the presence of aromatic DNA adducts in the biopsy samples at levels of up to 0.4 fmol total adducts/microgram DNA. Treatment of human skin in organ culture produced similar levels of adducts, while treatment with dithranol, a non-mutagenic therapeutic agent, resulted in chromatograms indistinguishable from those from untreated controls. In mouse skin, coal-tar ointment and juniper tar gave similar DNA adduct levels, with a similar time-course of removal: maximum levels (0.5 fmol/microgram DNA) at 24 h after the final treatment declined rapidly to 0.05 fmol/microgram at 7 d, thereafter declining slowly over the succeeding 25 d. However, while coal-tar ointment produced only very low levels of adducts in mouse lung (less than 0.03 fmol/microgram DNA), juniper tar produced adducts at a high level (0.7 fmol/microgram DNA) that were persistent in this tissue. These results provide direct evidence for the formation of potentially carcinogenic DNA damage in human and mouse tissue by components of these therapeutic tar preparations.

  9. Knee Adduction Moment and Medial Contact Force – Facts about Their Correlation during Gait

    PubMed Central

    Kutzner, Ines; Trepczynski, Adam; Heller, Markus O.; Bergmann, Georg

    2013-01-01

    The external knee adduction moment is considered a surrogate measure for the medial tibiofemoral contact force and is commonly used to quantify the load reducing effect of orthopedic interventions. However, only limited and controversial data exist about the correlation between adduction moment and medial force. The objective of this study was to examine whether the adduction moment is indeed a strong predictor for the medial force by determining their correlation during gait. Instrumented knee implants with telemetric data transmission were used to measure tibiofemoral contact forces in nine subjects. Gait analyses were performed simultaneously to the joint load measurements. Skeletal kinematics, as well as the ground reaction forces and inertial parameters, were used as inputs in an inverse dynamics approach to calculate the external knee adduction moment. Linear regression analysis was used to analyze the correlation between adduction moment and medial force for the whole stance phase and separately for the early and late stance phase. Whereas only moderate correlations between adduction moment and medial force were observed throughout the whole stance phase (R2 = 0.56) and during the late stance phase (R2 = 0.51), a high correlation was observed at the early stance phase (R2 = 0.76). Furthermore, the adduction moment was highly correlated to the medial force ratio throughout the whole stance phase (R2 = 0.75). These results suggest that the adduction moment is a surrogate measure, well-suited to predicting the medial force ratio throughout the whole stance phase or medial force during the early stance phase. However, particularly during the late stance phase, moderate correlations and high inter-individual variations revealed that the predictive value of the adduction moment is limited. Further analyses are necessary to examine whether a combination of other kinematic, kinetic or neuromuscular factors may lead to a more reliable prediction of

  10. Knee adduction moment and medial contact force--facts about their correlation during gait.

    PubMed

    Kutzner, Ines; Trepczynski, Adam; Heller, Markus O; Bergmann, Georg

    2013-01-01

    The external knee adduction moment is considered a surrogate measure for the medial tibiofemoral contact force and is commonly used to quantify the load reducing effect of orthopedic interventions. However, only limited and controversial data exist about the correlation between adduction moment and medial force. The objective of this study was to examine whether the adduction moment is indeed a strong predictor for the medial force by determining their correlation during gait. Instrumented knee implants with telemetric data transmission were used to measure tibiofemoral contact forces in nine subjects. Gait analyses were performed simultaneously to the joint load measurements. Skeletal kinematics, as well as the ground reaction forces and inertial parameters, were used as inputs in an inverse dynamics approach to calculate the external knee adduction moment. Linear regression analysis was used to analyze the correlation between adduction moment and medial force for the whole stance phase and separately for the early and late stance phase. Whereas only moderate correlations between adduction moment and medial force were observed throughout the whole stance phase (R(2) = 0.56) and during the late stance phase (R(2) = 0.51), a high correlation was observed at the early stance phase (R(2) = 0.76). Furthermore, the adduction moment was highly correlated to the medial force ratio throughout the whole stance phase (R(2) = 0.75). These results suggest that the adduction moment is a surrogate measure, well-suited to predicting the medial force ratio throughout the whole stance phase or medial force during the early stance phase. However, particularly during the late stance phase, moderate correlations and high inter-individual variations revealed that the predictive value of the adduction moment is limited. Further analyses are necessary to examine whether a combination of other kinematic, kinetic or neuromuscular factors may lead to a more reliable

  11. Polycyclic aromatic hydrocarbon-DNA adducts and survival among women with breast cancer

    SciTech Connect

    Sagiv, Sharon K. Gaudet, Mia M.; Eng, Sybil M.; Abrahamson, Page E.; Shantakumar, Sumitra; Teitelbaum, Susan L.; Bell, Paula; Thomas, Joyce A.; Neugut, Alfred I.; Santella, Regina M.; Gammon, Marilie D.

    2009-04-15

    Polycyclic aromatic hydrocarbons (PAH) are mammary carcinogens in animal studies, and a few epidemiologic studies have suggested a link between elevated levels of PAH-DNA adducts and breast cancer incidence. An association between PAH-DNA adducts and survival among breast cancer cases has not been previously reported. We conducted a survival analysis among women with newly diagnosed invasive breast cancer between 1996 and 1997, enrolled in the Long Island Breast Cancer Study Project. DNA was isolated from blood samples that were obtained from cases shortly after diagnosis and assayed for PAH-DNA adducts using ELISA. Among the 722 cases with PAH-DNA adduct measurements, 97 deaths (13.4%) from all causes and 54 deaths (7.5%) due to breast cancer were reported to National Death Index (NDI) by December 31, 2002. Using Cox proportional hazards models and controlling for age at diagnosis, we did not find evidence that all-cause mortality (hazard ratio (HR)=0.88; 95% confidence interval (CI): 0.57-1.37), or breast cancer mortality (HR=1.20; 95% CI: 0.63-2.28) was strongly associated with detectable PAH-DNA adduct levels compared with non-detectable adducts; additionally, no dose-response association was observed. Among a subgroup with treatment data (n=520), adducts were associated with over a two-fold higher mortality among those receiving radiation, but mortality for adducts was reduced among hormone therapy users. Results from this large population-based study do not provide strong support for an association between detectable PAH-DNA adducts and survival among women with breast cancer, except perhaps among those receiving radiation treatment.

  12. Relationship of methylglyoxal-adduct biogenesis to LDL and triglyceride levels in diabetics.

    PubMed

    Turk, Zdenka; Cavlović-Naglić, Maja; Turk, Nikša

    2011-09-26

    Protein glycation leading to advanced glycation-endproducts (AGE) is enhanced in diabetes by increased blood glucose and collateral endogenous production of reactive α-dicarbonyls. Among AGE precursors, methylglyoxal (MG) is considered as one of the key intermediates. We hypothesized it to be a common product of both carbonyl and oxidative stress, and investigated its biogenesis in relation to glycemic and lipid status in diabetic patients. Serum and urine MG-adducts were measured by competitive immunofluorometric assay in 83 diabetic and 20 healthy subjects. A significant association of MG-adducts serum level with LDL (r=0.31;p=0.003) was observed. A correlation between LDL-c, HDL-C and PPG as independent variables and serum MG-adducts as a dependent variable was found (p<0.014) using multiple stepwise regression, whereas urine albumin/creatinine ratio was independently associated with urine MG-adducts. LDL cut-off >3.0mmol/l discriminated patients with higher serum MG-adducts (p=0.0052), although there was no between-subgroup difference in glycemic control. Patients on statin therapy had a lower MG-adduct level. The positive relationship between LDL-c and MG-adducts (r=0.38;p=0.042) was noted in patients free of statin treatment, whereas an inverse tendency was found in the statin-treated subgroup. Significant relationship between LDL and MG-adduct production, as well as tight correlation between triglycerides and urinary MG-adduct excretion suggest that the lipoxidation and glyceraldehyde-3-phosphate route, along with the glycolytic pathway, might be an important source of MG generation. The glycotoxin methylglyoxal seems to be a common factor linking hyperglycemia and intensive lipolysis, two dominant metabolic changes in diabetes. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Mycotoxin Adducts on Human Serum Albumin: Biomarkers of Exposure to Stachybotrys chartarum

    PubMed Central

    Yike, Iwona; Distler, Anne M.; Ziady, Assem G.; Dearborn, Dorr G.

    2006-01-01

    Objective Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG)–albumin adducts may serve as biomarkers of exposure to this fungus. Design We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. Results Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine–, cysteine–, and histidine–SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. Conclusions These data document the occurrence of SG–albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. Relevance to clinical practice SG–amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum. PMID:16882529

  14. Mycotoxin adducts on human serum albumin: biomarkers of exposure to Stachybotrys chartarum.

    PubMed

    Yike, Iwona; Distler, Anne M; Ziady, Assem G; Dearborn, Dorr G

    2006-08-01

    Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG) -albumin adducts may serve as biomarkers of exposure to this fungus. We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine-, cysteine-, and histidine-SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. These data document the occurrence of SG-albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. SG-amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum.

  15. 7-Alkylguanine adduct levels in urine, lungs and liver of mice exposed to styrene by inhalation

    SciTech Connect

    Vodicka, Pavel Erik . E-mail: pvodicka@biomed.cas.cz; Linhart, Igor; Novak, Jan; Koskinen, Mikko; Vodickova, Ludmila; Hemminki, Kari

    2006-01-15

    This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7{alpha}G) and 7-(2-hydroxy-2-phenylethyl)guanine (N7{beta}G), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32 + 1.14 and 6.91 + 1.17 pmol/animal for lower and higher styrene exposure, respectively. {beta}-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F = 13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10{sup 8} normal nucleotides, i.e., 0.74 fmol/{mu}g DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m{sup 3}, while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing {alpha}-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0 x 10{sup -5}% of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly.

  16. Malondialdehyde-protein adducts in the spleens of aniline-treated rats: immunochemical detection and localization.

    PubMed

    Khan, M Firoze; Wu, X; Ansari, G A S; Boor, Paul J

    2003-01-10

    Previously it was reported that aniline exposure in rats induces increased lipid peroxidation and formation of malondialdehyde (MDA)-protein adducts in the spleen. In order to further elucidate the role of MDA-protein adducts in the splenic toxicity of aniline, studies were conducted to detect and localize these adducts in the spleen. Rabbit polyclonal antisera to MDA-keyhole limpet hemocyanin were employed for immunohistochemical localization and Western blot analyses of MDA-protein adducts in the spleens of aniline-treated (65 mg/kg/d aniline in the drinking water for 30 d) and control rats. For immunohistochemical localization of MDA-protein adducts in the spleen, a new approach using alkaline phosphatase-fast red (red color) to demonstrate bound primary antibodies was adopted, providing a sharper and increased contrast compared to horseradish peroxidase-diaminobenzidine (brown color) methodology. This new approach allowed us to differentiate the changes in aniline-treated spleens, which had extensive brownish deposits of iron proteins. Spleens from aniline-treated rats showed intense staining for these adducts in the red pulp areas (where iron was also localized), especially within the sinusoidal macrophages. Spleens from control rats showed only mild staining for adducts and only traces of iron. Western blot analyses of splenic microsomal proteins from aniline-treated and control rats showed the presence of 13 different MDA-modified proteins. However, 26-, 32-, and 14-kD proteins were more prominent in the aniline-treated rats. The colocalization of MDA-protein adducts with iron in the red pulp of the spleen suggests that iron-catalyzed lipid peroxidation leading to formation of MDA-protein adducts could be a potential mechanism for splenic toxicity of aniline.

  17. Hemoglobin and DNA adduct formation in Fischer-344 rats exposed to 2,4- and 2,6-toluene diamine.

    PubMed

    Wilson, P M; La, D K; Froines, J R

    1996-01-01

    Using gas chromatography/mass spectrometry for detection of hemoglobin adducts, and 32P-postlabelling for DNA adducts, we examined macromolecular binding in Fischer-344 rats administered 2,4-or 2,6-toluene diamine (TDA). The dose-response and correlative relationship between the two macromolecules were investigated over a range of doses (0-250 mg/kg). The time course of adduct formation and removal was also examined. Both TDA isomers induced formation of hemoglobin adducts, but only the 2,4-isomer induced DNA binding. Maximum hemoglobin and DNA adduct levels were detected 24 h following administration. Both hemoglobin and DNA binding increased in a dose-dependent manner. Hemoglobin adduct clearance demonstrated a nonlinear decay, with adduct loss occurring faster than normal erythrocyte clearance. The effects of metabolic inhibitors on adduct formation were examined using piperonyl butoxide and pentachlorophenol to inhibit p450 isozymes and sulfotransferase, respectively. Microsomal enzymatic activation was critical to hemoglobin adduct formation with inhibition by piperonyl butoxide reducing adduct yields by over 90%. Sulfation did not appear to play a significant role in TDA-induced hemoglobin adduct formation.

  18. Evolution of Research on the DNA Adduct Chemistry of N-Nitrosopyrrolidine and Related Aldehydes

    PubMed Central

    Hecht, Stephen S.; Upadhyaya, Pramod; Wang, Mingyao

    2011-01-01

    This perspective reviews our work on the identification of DNA adducts of N-nitrosopyrrolidine and some related aldehydes. The research began as a focused project to investigate mechanisms of cyclic nitrosamine carcinogenesis but expanded into other areas as aldehyde metabolites of NPYR were shown to have their own diverse DNA adduct chemistry. A total of 69 structurally distinct DNA adducts were identified and some of these, found in human tissues, have provided intriguing leads for investigating carcinogenesis mechanisms in humans due to exposure to both endogenous and exogenous agents. PMID:21480629

  19. Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct.

    PubMed

    Politica, Dustin A; Malik, Chanchal K; Basu, Ashis K; Stone, Michael P

    2015-12-21

    3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-d

  20. DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes

    PubMed Central

    Nauwelaers, Gwendoline; Bessette, Erin E.; Gu, Dan; Tang, Yijin; Rageul, Julie; Fessard, Valérie; Yuan, Jian-Min; Yu, Mimi C.; Langouët, Sophie; Turesky, Robert J.

    2011-01-01

    DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 107 DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation. PMID:21456541

  1. DNA adducts as a measure of lung cancer risk in humans exposed to polycyclic aromatic hydrocarbons

    SciTech Connect

    Kriek, E.; Van Schooten, F.J.; Hillebrand, M.J.X.; Van Leeuwen, F.E.; Den Engelse, L.; De Looff, A.J.A.; Dijkmans, A.P.G.

    1993-03-01

    Workers in the coking, foundry, and aluminum industry can be exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and are at increased risk for lung cancer, as are cigarette smokers. In recent years several studies on workers in the foundry and coking industries have been reported. In these studies, white blood cell (WBC) DNA was used for analysis of PAH-DNA adducts. Theoretically, DNA adduct formation is a more relevant biological parameter for assessing exposure risk than PAH in the work atmosphere, or the amount of a metabolite in the urine, because adduct levels reflect that part of the dose that escapes detoxification and binds to DNA. WBC DNA was analyzed from coke-oven workers and from workers in an aluminum production plant and demonstrated the presence of PAH-DNA adducts. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their WBC compared with 27% of the controls (p < 0.05), measured with ELISA. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. In the aluminum workers, no PAH-DNA adducts were detected by ELISA, although the benzo[a]pyrene concentrations in the work atmosphere were comparable to those of the coke-oven workers. The more sensitive [sup 32]P-postlabeling assay showed the presence of PAH-DNA adducts in 91% of the aluminum workers. There was no correlation of WBC adduct levels with the concentration of PAH in the work atmosphere. Recently the authors showed that total PAH-DNA adduct levels in WBC from lung cancer patients were much higher than those generally found in healthy smokers. These increased adduct levels may indicate a subpopulation of smokers with increased risk for lung cancer, resulting from a genetic predisposition in this group of persons. Because WBCs are not the target cells for exposure-related cancer, the relationship between PAH-DNA adducts in the lung and in WBCs remains to be established. 30 refs., 2 figs., 2 tabs.

  2. Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53

    NASA Astrophysics Data System (ADS)

    Denissenko, Mikhail F.; Pao, Annie; Tang, Moon-Shong; Pfeifer, Gerd P.

    1996-10-01

    Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

  3. The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

    PubMed Central

    Chakravarti, Dhrubajyoti; Venugopal, Divya; Mailander, Paula C.; Meza, Jane L.; Higginbotham, Sheila; Cavalieri, Ercole L.; Rogan, Eleanor G.

    2008-01-01

    Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N2dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early

  4. Preoperative Concurrent Radiation Therapy and Chemotherapy for Bulky Stage IB2, IIA, and IIB Carcinoma of the Uterine Cervix With Proximal Parametrial Invasion

    SciTech Connect

    Huguet, Florence; Cojocariu, Oana-Maria; Levy, Pierre; Lefranc, Jean-Pierre; Darai, Emile; Jannet, Denis; Ansquer, Yan; Lhuillier, Pierre-Eugene; Benifla, Jean-Louis; Seince, Nathalie; Touboul, Emmanuel

    2008-12-01

    Purpose: To evaluate toxicity, local tumor control, and survival after preoperative chemoradiation for operable bulky cervical carcinoma. Methods and Materials: Between December 1991 and July 2006, 92 patients with operable bulky stage IB2, IIA, and IIB cervical carcinoma without pelvic or para-aortic nodes on pretreatment imaging were treated. Treatment consisted of preoperative external beam pelvic radiation therapy (EBRT) and concomitant chemotherapy (CT) during the first and fourth weeks of radiation combining 5-fluorouracil and cisplatin. The pelvic radiation dose was 40.5 Gy over 4.5 weeks. EBRT was followed by low-dose rate uterovaginal brachytherapy with a total dose of 20 Gy in 62 patients. After a median rest period of 44 days, all patients underwent Class II modified radical hysterectomy with bilateral pelvic lymphadenectomy. Thirty patients who had not received preoperative uterovaginal brachytherapy underwent postoperative low-dose-rate vaginal brachytherapy at a dose of 20 Gy. The mean follow-up was 46 months. Results: Pathologic residual tumor was observed in 43 patients. After multivariate analysis, additional preoperative uterovaginal brachytherapy was the single significant predictive factor for pathologic complete response rate (p = 0.019). The 2- and 5-year disease-free survival (DFS) rates were 80.4% and 72.2%, respectively. Pathologic residual cervical tumor was the single independent factor decreasing the probability of DFS (p = 0.020). Acute toxicities were moderate. Two severe ureteral complications requiring surgical intervention were observed. Conclusions: Concomitant chemoradiation followed by surgery for operable bulky stage I-II cervical carcinoma without clinical lymph node involvement can be used with acceptable toxicity. Pathologic complete response increases the probability of DFS.

  5. N-Heterocyclic Carbene-Carbodiimide ("NHC-CDI") Adduct or Zwitterionic-Type Neutral Amidinate-Supported Magnesium(II) and Zinc(II) Complexes.

    PubMed

    Baishya, Ashim; Kumar, Lokesh; Barman, Milan Kr; Biswal, Himansu S; Nembenna, Sharanappa

    2017-08-21

    A series of structurally characterized magnesium and zinc complexes of the form L(4-tBuPh)-M{N(SiMe3)2}2 [M = Mg (1) and Zn (2); L(4-tBuPh) = 1,3-diethyl-4,5-dimethylimidazolium-2-{N,N'-bis(4-tert-butylphenyl)amidinate}], L(4-iPrPh)-M{N(SiMe3)2}2 [M = Mg (3) and Zn (4); L(4-iPrPh) = 1,3-diethyl-4,5-dimethylimidazolium-2-{N,N'-bis(4-isopropylphenyl)amidinate}], and L(4-iPrPh)-ZnEt2 (5) bearing a zwitterionic-type neutral amidinate or N-heterocyclic carbene-carbodiimide ("NHC-CDI") adduct and monoanionic amido or alkyl ligands have been reported. The synthesis of compounds 1-5 was achieved by the direct addition of a "NHC-CDI" adduct to a corresponding metal bis(amide) or dialkyl reagent. All compounds 1-5 exist as monomers in the solid state. In all cases, the metal (magnesium or zinc) centers adopt a distorted four-coordinate tetrahedral geometry bonded to one N,N'-chelated neutral zwitterionic ligand and two monoanionic amido or alkyl moieties. In contrast, sterically bulky zwitterionic amidinate 1,3-diethyl-4,5-dimethylimidazolium-2-{N,N'-bis(2,6-diisopropylphenyl)amidinate} (L(Dipp)) upon treatment with lithium bis[(trimethylsilyl)amide], Li{N(SiMe3)2}, affords the NHC-lithium complex (Me)IEt-[Li{N(SiMe3)2}]2 (6), in which one molecule of NHC ((Me)IEt = 1,3-diethyl-4,5-dimethylimidazol-2-ylidene) coordinates to one of the two lithium centers. In a similar way, the reaction between L(Dipp) and Mg{N(SiMe3)2}2 allowed the formation of a NHC adduct of metal bis(amide), (Me)IEt-Mg{N(SiMe3)2}2 (7), instead of a zwitterionic adduct of metal bis(amide). Alternatively, the synthesis of both compounds 6 and 7 was achieved by the direct addition of 1 equiv of NHC, i.e., (Me)IEt to Li{N(SiMe3)2} (2.0 equiv) and Mg{N(SiMe3)2}2 (1.0 equiv) in benzene-d6, respectively. All compounds (1-7) were characterized by multinuclear {(1)H, (13)C, and (29)Si (for 1-4, 6, and 7) and (7)Li (for compound 6)} magnetic resonance spectroscopy, mass spectrometry, elemental analysis, and single

  6. Unusual mixed valent FeIIIFeII complex (St = 3/2) stabilised by a reduced bulky 1,2-diketone.

    PubMed

    Spikes, Geoffrey H; Bill, Eckhard; Weyhermüller, Thomas; Wieghardt, Karl

    2007-11-14

    The reduction of the bulky 1,2-diketone bis(2,6-diisopropylphenyl)glyoxal () and FeBr(2) with 1.5 equivalents of Na results in a Class 2 mixed valent H.S. Fe(II) L.S. Fe(III) complex (2) with two five-coordinate Fe centres which are antiferromagnetically exchange-coupled to give a total spin S(t) = 3/2 ground state and an S(t) = 5/2 excited state that are separated by about 25 cm(-1) (for Delta(J) approximately 5J).

  7. Identification and characterization of the major DNA adduct formed chemically and in vitro from the environmental genotoxin 3-nitrofluoranthene.

    PubMed

    Dietrich, A M; Guenat, C R; Tomer, K B; Ball, L M

    1988-11-01

    The genotoxic environmental pollutant 3-nitrofluoranthene (3-NFA) was reduced chemically and allowed to react with calf thymus DNA, yielding one major adduct which was determined to be N-(deoxyguanosin-8-yl)-3-amino-fluoranthene based on Fast Atom Bombardment Mass Spectrometry (FAB-MS), proton nuclear magnetic resonance, ultraviolet-visible wavelength light spectroscopy (UV-VIS), and fluorescence data. Extensive characterization of the isolated adduct by tandem mass spectrometry (MS/MS) was necessary to demonstrate definitively that the adduct isolated was the dG:C8 adduct, and not the isomeric dG:N2 adduct. The extent of modification of the initial calf thymus DNA by chemically reduced 3-NFA was 0.12% (1.2 adducts/10(3) nucleosides), which was sufficient to allow several hundred micrograms of the adduct to be isolated and purified. The chemically synthesized adduct was utilized as a reference standard for comparison to the major adduct isolated from xanthine-oxidase-catalyzed reduction of 3-NFA in vitro. The yield from the in vitro biological system was 2.4 adducts/10(5) nucleosides; the adduct isolated possessed the same mass spectrometric, UV-VIS, and fluorescence characteristics as the purified standard, and co-eluted with the standard on HPLC. No evidence for other adducts was found, either in vitro or in the chemical synthesis, based on FAB-MS examination of whole extracts of the reaction mixture for the presence of ions related to other possible adducts. Therefore, if minor adducts were present they were formed in substantially lesser amounts than N-(deoxyguanosin-8-yl)-3-aminofluoranthene.

  8. Formation, persistence, and identification of DNA adducts formed by the carcinogenic environmental pollutant o-anisidine in rats.

    PubMed

    Naiman, Karel; Dracínský, Martin; Hodek, Petr; Martínková, Markéta; Schmeiser, Heinz H; Frei, Eva; Stiborová, Marie

    2012-06-01

    2-Methoxyaniline (o-anisidine) is an industrial and environmental pollutant causing tumors of urinary bladder in rodents. Here, we investigated the formation and persistence of DNA adducts in the Wistar rat. Using the (32)P-postlabeling method, three o-anisidine-derived DNA adducts were found in several organs of rats treated with a total dose of 0.53 mg o-anisidine/kg body wt (0.15, 0.18, and 0.2 mg/kg body wt ip in the first, second, and third day, respectively), of which the urinary bladder had the highest levels. At four posttreatment times (1 day, 13 days, 10 weeks, and 36 weeks), DNA adducts in bladder, liver, kidney, and spleen of rats were analyzed to study their persistence. In all time points, the highest total adduct levels were found in urinary bladder (39 adducts per 10(7) nucleotides after 1 day and 15 adducts per 10(7) nucleotides after 36 weeks) where 39% adducts remained. In contrast to the urinary bladder, no persistence was detected in other organs. All three DNA adducts were identified as deoxyguanosine adducts. When deoxyguanosine was reacted with the oxidative metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine, three adducts could be separated by high-performance liquid chromatography (HPLC) and were identified by mass spectroscopy and/or nuclear magnetic resonance spectrometry. All adducts are products of the nitrenium/carbenium ions, the reactive species generated from N-(2-methoxyphenyl)hydroxylamine. The major adduct was identified to be N-(deoxyguanosin-8-yl)-2-methoxyaniline. Using cochromatography on HPLC, this adduct was found to be identical to the major adduct generated by activation of o-anisidine in vitro and in vivo.

  9. Acetaminophen-induced liver injury in rats and mice: Comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity

    SciTech Connect

    McGill, Mitchell R.; Williams, C. David; Xie, Yuchao; Ramachandran, Anup; Jaeschke, Hartmut

    2012-11-01

    Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity. Highlights: ► Acetaminophen overdose causes severe liver injury only in mice but not in rats. ► APAP causes hepatic GSH depletion and protein adduct formation in rats and mice. ► Less protein adducts were measured in rat liver mitochondria compared to mouse. ► No oxidant stress, peroxynitrite formation or JNK activation was present in rats. ► The

  10. Methods for synthesizing alane without the formation of adducts and free of halides

    DOEpatents

    Zidan, Ragaiy; Knight, Douglas A; Dinh, Long V

    2013-02-19

    A process is provided to synthesize an alane without the formation of alane adducts as a precursor. The resulting product is a crystallized .alpha.-alane and is a highly stable product and is free of halides.

  11. Stability and proton transfer in DNA base pairs of AMD473-DNA adduct

    NASA Astrophysics Data System (ADS)

    Sarmah, Pubalee; Deka, Ramesh C.

    2011-05-01

    We investigate the energetics of four different adducts of cisplatin analogue cis-[PtCl 2(NH 3)(2-picoline)] (AMD473) with a duplex DNA using DFT/ONIOM methods to probe their stabilities. Further, we study the possibilities of proton transfer between DNA base pairs of the most stable drug-DNA adduct. The adduct b(2-picoline trans to 3'-G and 2-methyl group directs to the DNA major groove) is found to be the most stable configuration among all the possible adducts. From the proton transfer analysis we found that the single proton transfer between N1 position of guanine (G) and N3 position of cytosine (C) of each GC pair gives a structure energetically as stable as the original one.

  12. Lifetimes and stabilities of familiar explosive molecular adduct complexes during ion mobility measurements.

    PubMed

    McKenzie-Coe, Alan; DeBord, John Daniel; Ridgeway, Mark; Park, Melvin; Eiceman, Gary; Fernandez-Lima, Francisco

    2015-08-21

    Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) was utilized for the separation and identification of familiar explosives in complex mixtures. For the first time, molecular adduct complex lifetimes, relative stability, binding energies and candidate structures are reported for familiar explosives. Experimental and theoretical results showed that the adduct size and reactivity, complex binding energy and the explosive structure tailor the stability of the molecular adduct complex. The flexibility of TIMS to adapt the mobility separation as a function of the molecular adduct complex stability (i.e., short or long IMS experiments/low or high IMS resolution) permits targeted measurements of explosives in complex mixtures with high confidence levels.

  13. Direct alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl adducts of guanine and thymine and carboxyethyl adducts of adenine and cytosine.

    PubMed Central

    Solomon, J J; Segal, A

    1985-01-01

    Reaction of the rodent carcinogen acrylonitrile (AN) at pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. The carboxyethyl adducts resulted from initial cyanoethylation at a ring nitrogen adjacent to an exocyclic nitrogen followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is the result of an intramolecular-catalyzed reaction resulting from the formation of a transient cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated was 1-CE-Ade (25.8%), N6-CE-Ade (7.6%), 3-CE-Cyt (1.3%), 7-CNE-Gua (25.8%), 7,9-bis-CNE-Gua (4.3%), imidazole ring-opened 7,9-bis-CNE-Gua (18.9%) and 3-CNE-Thy (16.3%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4085427

  14. Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from a Drug Acyl Glucuronide Metabolite: Multiple Albumin Adductions in Diclofenac Patients

    PubMed Central

    Hammond, Thomas G.; Meng, Xiaoli; Jenkins, Rosalind E.; Maggs, James L.; Castelazo, Anahi Santoyo; Regan, Sophie L.; Bennett, Stuart N. L.; Earnshaw, Caroline J.; Aithal, Guruprasad P.; Pande, Ira; Kenna, J. Gerry; Stachulski, Andrew V.; Park, B. Kevin

    2014-01-01

    Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-related hypersensitivities had either a single drug-derived adduct or one of five combinations of 2–8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein’s 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug’s AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association. PMID:24902585

  15. Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring*

    PubMed Central

    Li, He; Grigoryan, Hasmik; Funk, William E.; Lu, Sixin Samantha; Rose, Sherri; Williams, Evan R.; Rappaport, Stephen M.

    2011-01-01

    A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. PMID:21193536

  16. Adduction of the chloroform metabolite phosgene to lysine residues of human histone H2B.

    PubMed

    Fabrizi, Laura; Taylor, Graham W; Cañas, Benito; Boobis, Alan R; Edwards, Robert J

    2003-03-01

    Human exposure to trihalomethanes such as chloroform has been associated with both cancer and reproductive toxicity. While there is little evidence for chloroform mutagenicity or DNA adduct formation, in vivo studies in rats have demonstrated adduction to histones and other nuclear proteins. Histones play a key role in controlling DNA expression particularly through the acetylation of lysine residues in their N-termini. Therefore, we studied the reaction of phosgene, the major active metabolite of chloroform, with the N-terminus of human histone H2B (Hpep, Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys) in a model chemical system. The aim of this study was to assess whether phosgene is able to form irreversible adducts with this peptide and to investigate which residues are most susceptible. Hpep was reacted with a range of phosgene concentrations (0.03-36 mM) at 37 degrees C, pH 7.4. The products of these reactions, analyzed by matrix-assisted laser desorption ionization MS, showed that up to three CO moieties could be adducted to the peptide. The singly and doubly adducted peptides were purified by HPLC and then hydrolyzed with trypsin to produce a series of fragments that were analyzed by HPLC-MS. The tryptic products showed that adduction occurred principally at lysine residues, and that all seven lysine residues of the peptide were subject to adduction. Collision-induced dissociation analysis using ion trap MS-MS of the tryptic fragment [Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys + CO] and of the full-length singly adducted peptide supported the role of lysine residues in adduction; the data also indicated that the N-terminal proline and the serine residues are susceptible. Addition of glutathione to the reaction mixture only partially attenuated adduct formation and allowed production of another adducted species, i.e., Hpep-CO-glutathione. The occurrence of such reactions to the N-termini of histones, if confirmed

  17. NMR solution structures of adducts derived from the binding of polycyclic aromatic diol epoxides to DNA

    SciTech Connect

    Cosman, M.; Patel, D.J.; Hingerty, B.E.; Amin, S.; Broyde, S.; Geacintov, N.E.

    1995-12-31

    Site-specifically modified oligonucleotides were derived from the reactions of stereoisomeric polycyclic aromatic diol epoxide metabolite model compounds with oligonucleotides of defined base composition and sequence. The NMR solution structures of ten different adducts studied so far are briefly described, and it is shown that stereochemical factors and the nature of the oligonucleotide context of the complementary strands, exert a powerful influence on the conformational features of these adducts.

  18. Activation of DNA damage response pathways as a consequence of anthracycline-DNA adduct formation.

    PubMed

    Forrest, Robert A; Swift, Lonnie P; Rephaeli, Ada; Nudelman, Abraham; Kimura, Ken-Ichi; Phillips, Don R; Cutts, Suzanne M

    2012-06-15

    The cytotoxicity of doxorubicin, a clinically used anti-neoplastic drug, can be enhanced by formaldehyde (either endogenous or exogenous) to promote the formation of doxorubicin-DNA adducts. Formaldehyde supplies the carbon required for the covalent linkage of doxorubicin to one strand of DNA, with hydrogen bonds stabilising the doxorubicin mono-adduct to the other strand of DNA, to act much like an interstrand crosslink. Interstrand crosslinks present a major challenge for cellular repair processes, requiring the activation of numerous DNA damage response proteins for resolution of the resulting DNA intermediates and damage. This work investigates DNA damage response proteins activated by doxorubicin-DNA adducts. Although p53 was phosphorylated at Serine 15 in response to adducts, long term growth inhibition of mammalian cells was not affected by p53 status. Using siRNA technology and kinase inhibitors we observed enhanced cellular sensitivity to doxorubicin-DNA adducts when the activity of the signalling protein kinases ATM and ATR were lost. Cells synchronised using a double thymidine block were sensitised to adduct-initiated cell death upon ATR knockdown, but relatively unaffected by ATM knockdown. Loss of ATR was associated with abrogation of a drug-induced G(2)/M block and induction of mitotic catastrophe, while loss of ATM was associated with drug-induced apoptosis in non-synchronised cells. These proteins may therefore be potential drug targets to achieve synergistic cytotoxic responses to doxorubicin-DNA adduct forming therapies. The analysis of these protein kinases with respect to cell cycle progression indicates that ATR is required for G(2)/M checkpoint responses while ATM appears to function in G(1) mediated responses to anthracycline adducts. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Synthesis of a major mitomycin C DNA adduct via a triaminomitosene.

    PubMed

    Champeil, Elise; Paz, Manuel M; Lukasiewicz, Elaan; Kong, Wan S; Watson, Stephanie; Sapse, Anne-Marie

    2012-12-01

    We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O(6)-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N(2) and O(6) positions.

  20. Tamoxifen–DNA adduct formation in monkey and human reproductive organs

    PubMed Central

    Hernandez-Ramon, Elena E.

    2014-01-01

    The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM–DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM–DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3–4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM–DNA adducts were measurable by TAM–DNA chemiluminescence immunoassay. Average TAM–DNA adduct values for the patas uteri (23 adducts/108 nucleotides) were similar to those found in endometrium of the macaques (19 adducts/108 nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/108 nucleotides. To examine TAM–DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM–DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM–DNA adducts/108 nucleotides, whereas unexposed women showed no detectable TAM–DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer. PMID:24501327

  1. Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells

    PubMed Central

    Berger, John P.; Simet, Samantha M.; DeVasure, Jane M.; Boten, Jessica A.; Sweeter, Jenea M.; Kharbanda, Kusum K.; Sisson, Joseph H.; Wyatt, Todd A.

    2014-01-01

    Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3–7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3–7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium. PMID:24880893

  2. Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells.

    PubMed

    Berger, John P; Simet, Samantha M; DeVasure, Jane M; Boten, Jessica A; Sweeter, Jenea M; Kharbanda, Kusum K; Sisson, Joseph H; Wyatt, Todd A

    2014-08-01

    Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3-7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3 to 7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.

  3. Oxidation and glycolytic cleavage of etheno and propano DNA base adducts.

    PubMed

    Knutson, Charles G; Rubinson, Emily H; Akingbade, Dapo; Anderson, Carolyn S; Stec, Donald F; Petrova, Katya V; Kozekov, Ivan D; Guengerich, F Peter; Rizzo, Carmelo J; Marnett, Lawrence J

    2009-02-03

    Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2'-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N(2)-epsilon-guanine (1,N(2)-epsilon-Gua), N(2),3-epsilon-Gua, heptanone-1,N(2)-epsilon-Gua, 1,N(6)-epsilon-adenine (1,N(6)-epsilon-Ade), and 3,N(4)-epsilon-cytosine (3,N(4)-epsilon-Cyt)] and their corresponding 2'-deoxynucleosides. Both 1,N(2)-epsilon-Gua and heptanone-1,N(2)-epsilon-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M(1)G and 1,N(2)-propanoguanine (PGua)] and their 2'-deoxynucleoside derivatives were not oxidized. The 2'-deoxynucleoside adducts, 1,N(2)-epsilon-dG and 1,N(6)-epsilon-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransformation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies.

  4. Factors influencing knee adduction moment measurement: A systematic review and meta-regression analysis.

    PubMed

    Telfer, Scott; Lange, Moritz J; Sudduth, Amanda S M

    2017-08-24

    The external knee adduction moment has been identified as a key biomarker in biomechanics research, with associations with this variable and degenerative diseases such as knee osteoarthritis. Heterogeneity in participant characteristics and the protocols used to measure this variable may however complicate its interpretation. Previous reviews have focused on interventions or did not control for potential moderator variables in their analysis. In this meta-regression analysis, we aimed to determine the influence of factors including the cohort type, footwear, and walking speed on the measurement of knee adduction moment. We performed a systematic review of the literature, identifying articles that used the Plug-in-Gait inverse dynamics model to calculate the knee adduction moment during level walking, and used a mixed effect model to determine the effect of the previously described factors on the measurement. Results for 861 individuals were described in 19 articles. Walking speed had the largest influence on knee adduction moment (p<0.001), and participants with medial knee osteoarthritis had an increased knee adduction moment (p=0.008) compared to healthy subjects. Footwear was found to have a significant overall effect (p=0.024). Participants tested barefoot or wearing their own shoes had lower adduction moments than those tested in footwear provided by the researchers. Overall, the moderators accounted for 60% of the heterogeneity in the results. These results support the hypothesis that an increased knee adduction moment is associated with medial compartment knee osteoarthritis, and that footwear choice can influence the results. Gait speed has the largest effect on knee adduction moment measurement and should be carefully controlled for in studies investigating this variable. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Structural Elucidation of a Carnosine-Acrolein Adduct and its Quantification in Human Urine Samples.

    PubMed

    Bispo, Vanderson S; de Arruda Campos, Ivan P; Di Mascio, Paolo; Medeiros, Marisa H G

    2016-01-19

    Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic strategies for carnosine.

  6. Structural Elucidation of a Carnosine-Acrolein Adduct and its Quantification in Human Urine Samples

    PubMed Central

    Bispo, Vanderson S.; de Arruda Campos, Ivan P.; Di Mascio, Paolo; Medeiros, Marisa H. G.

    2016-01-01

    Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic strategies for carnosine. PMID:26783107

  7. Metabolic activation and DNA-adducts detection as biomarkers of chlorinated pesticide exposures.

    PubMed

    M Dubois Y Grosse J P Thome P Kremers And A Pfohl-Leszkowicz

    1997-01-01

    Several authors have reported the high hepatic incidence of γhexachlorocyclohexane (γHCH), pentachlorophenol (PCP) and hexachlorobenzene (HCB) which are widely used as pesticides. Their genotoxicity status was not clearly known and no m utagenic effects, using the Salmonella assay, were reported. In the first part of this report, DNA-adduct form ation is evaluated in three types of cultured hepatic cells (rodent, bird and hum an) as a biomarker of exposure to genotoxic com pounds. γHCH-, PCP- and HCB-DNA adducts were analysed, using the sensitive (32)P-postlabelling assay in its nuclease P1 enrichm ent version. The genotoxicity of lindane and PCP is clearly established. Total DNA-adducts reached a m axim um in foetal rat hepatocytes (17 and 15 adducts per 10(9) nucleotides) after an exposure to pentachlorophenol and lindane respectively. After HCB treatm ent, lim ited am ounts of DNA-adducts were found in the different cells used. The finding that DNA adducts were not the sam e in all species tested m ight be due to m etabolic differences. Each type of cultured cells preferentially express different cytochrome P450 fam ilies. These P450s m etabolize a wide variety of xenobiotics and bioactivate carcinogens into reactive m etabolites able to form DNA-adducts. The objective of the present study was to exam ine the possible association between DNA-adduction and particular C YP450 induction. The induced cytochrom e P450s were m easured by northern blot analysis. In rat and hum an cells, lindane treatm ent strongly induces CYP2B and CYP3A m RNA levels, whereas pentachlorophenol treatm ent induces CYP1A, CYP2B and CYP3A.

  8. Correlation of haemoglobin-acrylamide adducts with airborne exposure: an occupational survey.

    PubMed

    Jones, Kate; Garfitt, Sarah; Emms, Vicky; Warren, Nick; Cocker, John; Farmer, Peter

    2006-04-10

    This paper reports an occupational hygiene survey of exposure to acrylamide comparing acrylamide haemoglobin adduct measurements with personal air monitoring and glove liner analysis. The air monitoring data showed that exposure to acrylamide was well-controlled with all samples below the UK maximum exposure limit (MEL) of 300 microg/m(3) with mean exposure about one tenth of the MEL. Each worker provided two blood samples approximately 3 months apart. These samples were well correlated (r=0.61) with a slope of 0.74, indicating that exposure was reasonably constant. Mean personal airborne acrylamide levels and mean acrylamide haemoglobin adduct levels were well correlated (r=0.72, N=46) and using the calculated linear correlation, exposure at the MEL would be expected to give rise to a haemoglobin adduct level of 1,550 pmol/g globin. Smoking status did not affect the correlation. There was also a correlation between levels of acrylamide detected on gloves and haemoglobin adduct levels. A combined regression model between haemoglobin adducts, airborne acrylamide and acrylamide glove contamination was significant for both airborne acrylamide and gloves with a regression coefficient of 0.89. The study showed that haemoglobin adduct level was a good biomarker of acrylamide exposure which correlated to both inhaled and potentially skin absorbed acrylamide estimates. There was excellent discrimination between well-controlled occupational levels and environmental levels from diet and smoking, allowing haemoglobin adduct measurement to be used to determine even low level exposures. Due to the complexity of the current methodology, new techniques would be useful in making haemoglobin adducts more widely applicable.

  9. Tamoxifen-DNA adduct formation in monkey and human reproductive organs.

    PubMed

    Hernandez-Ramon, Elena E; Sandoval, Nicole A; John, Kaarthik; Cline, J Mark; Wood, Charles E; Woodward, Ruth A; Poirier, Miriam C

    2014-05-01

    The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.

  10. Passive limitation of adduction after Cüppers's 'Fadenoperation' on medial recti.

    PubMed Central

    Paliaga, G P; Braga, M

    1989-01-01

    In 40 eyes of 20 esotropic subjects in which a 'Fadenoperation' was performed on the medial recti we measured the resistance to ocular rotation in adduction before and after the operation. The difference between the two sets of force measurements demonstrates that the Fadenoperation on medial recti produces a mechanical restriction to adduction which can explain the effect of the surgical procedure on the strabismic deviation. PMID:2765442

  11. Dosimetry of Exposure to Sulfur Mustard of Human Skin: Immunofluorescence Microscopy of DNA-Adducts.

    DTIC Science & Technology

    used to develop methods for dosimetry by immunofluorescence microscopy of adducts to DNA after exposure to HD. This technique might be of great help...distinguished by cell-type specific immunochemical staining. Such techniques will facilitate investigations on the healing of skin injuries....recent years, monoclonal antibodies have been raised against a major HD-DNA adduct , i.e. N7-(2’-hydroxyethylthioethyl)-guanine. These antibodies have been

  12. Consolidation Radiotherapy in Stage IE- IIE, Non-Bulky Primary Gastric Diffuse Large B-Cell Lymphoma with Post-Chemotherapy Complete Remission.

    PubMed

    Li, Qiwen; Li, Wei; Wang, Liang; Wang, Weida; Niu, Shaoqing; Bi, Xiwen; Wang, Hanyu; Zhang, Yujing

    2015-01-01

    To investigate the effects of consolidation radiation in patients with stage IE-IIE, non-bulky primary gastric diffuse large B-cell lymphoma (DLBCL). A cohort consisted of 71 consecutive patients with stage IE-IIE, non-bulky primary gastric DLBCL was retrospectively analyzed. All of them had been in complete remission after receiving at least four cycles of chemotherapy, containing rituximab or not. Consolidation radiation was delivered thereafter in 28 patients while other 43 received clinical observation only. Locoregional relapse-free survival (LRFS), disease-free survival (DFS), overall survival (OS) and distant metastasis-free survival (DMFS) were compared between patients with or without radiotherapy. The 10-year LRFS, DFS, OS and DMFS were 100% and 81.4% (p = 0.028), 91.7% and 77.1% (p = 0.14), 91.7% and 77.8% (p = 0.67), 91.7% and 78.0% (p = 0.42) for patients with or without radiotherapy. Radiotherapy is associated with improved locoregional control of patients with early stage primary gastric DLBCL, who have achieved complete remission following at least four cycles of chemotherapy.

  13. Dose-response and failure pattern for bulky or barrel-shaped stage IB cervical cancer treated by combined photon irradiation and extrafascial hysterectomy

    SciTech Connect

    Maruyama, Y.; van Nagell, J.R.; Yoneda, J.; Donaldson, E.; Gallion, H.H.; Higgins, R.; Powell, D.; Kryscio, R.; Berner, B.

    1989-01-01

    From 1975 to 1987, 80 patients with bulky or barrel-shaped Stage IB cervical cancer were treated with preoperative irradiation and Cs-137 intracavitary implant therapy, before a planned extrafascial abdominal hysterectomy, using a consistent treatment policy. Of the hysterectomy specimens obtained, 37% were positive histologically at 89 +/- 2.3 days after the start of radiotherapy and at 4 to 6 weeks after the completion of radiation therapy. Sixty-three percent were negative after a total external and internal cervix irradiation dose of 9642 cGy at point T. The average point A dose contributed by intracavitary therapy was 2104 cGy. The survival rate at 5 years was 84%: At 10 years the survival rate was 78%. The failure pattern was analyzed for patients who had positive and negative specimens. The patients with positive specimens failed pelvically or pelvically and distantly. Patients with negative specimens failed in extrapelvic or distant metastatic sites. Preoperative radiotherapy led to excellent local and pelvic control of tumor, and the failures became predominantly distant metastases. The combined radiosurgical therapy was tolerated well and allowed surgical staging of disease. This permitted earlier and selective consideration of adjunctive therapy (i.e., paraaortic irradiation, chemotherapy, or chemoradiotherapy). The dose-response data give insight into the effects of photon radiotherapy on bulky or barrel Stage IB cervical cancers and correlate histologic status with failure pattern, outcome, and long-term survival.

  14. Structural Diversity in Alkali Metal and Alkali Metal Magnesiate Chemistry of the Bulky 2,6-Diisopropyl-N-(trimethylsilyl)anilino Ligand.

    PubMed

    Fuentes, M Ángeles; Zabala, Andoni; Kennedy, Alan R; Mulvey, Robert E

    2016-10-10

    Bulky amido ligands are precious in s-block chemistry, since they can implant complementary strong basic and weak nucleophilic properties within compounds. Recent work has shown the pivotal importance of the base structure with enhancement of basicity and extraordinary regioselectivities possible for cyclic alkali metal magnesiates containing mixed n-butyl/amido ligand sets. This work advances alkali metal and alkali metal magnesiate chemistry of the bulky arylsilyl amido ligand [N(SiMe3 )(Dipp)](-) (Dipp=2,6-iPr2 -C6 H3 ). Infinite chain structures of the parent sodium and potassium amides are disclosed, adding to the few known crystallographically characterised unsolvated s-block metal amides. Solvation by N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) or N,N,N',N'-tetramethylethylenediamine (TMEDA) gives molecular variants of the lithium and sodium amides; whereas for potassium, PMDETA gives a molecular structure, TMEDA affords a novel, hemi-solvated infinite chain. Crystal structures of the first magnesiate examples of this amide in [MMg{N(SiMe3 )(Dipp)}2 (μ-nBu)]∞ (M=Na or K) are also revealed, though these breakdown to their homometallic components in donor solvents as revealed through NMR and DOSY studies. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  15. Structural Diversity in Alkali Metal and Alkali Metal Magnesiate Chemistry of the Bulky 2,6‐Diisopropyl‐N‐(trimethylsilyl)anilino Ligand

    PubMed Central

    Fuentes, M. Ángeles; Zabala, Andoni; Kennedy, Alan R.

    2016-01-01

    Abstract Bulky amido ligands are precious in s‐block chemistry, since they can implant complementary strong basic and weak nucleophilic properties within compounds. Recent work has shown the pivotal importance of the base structure with enhancement of basicity and extraordinary regioselectivities possible for cyclic alkali metal magnesiates containing mixed n‐butyl/amido ligand sets. This work advances alkali metal and alkali metal magnesiate chemistry of the bulky arylsilyl amido ligand [N(SiMe3)(Dipp)]− (Dipp=2,6‐iPr2‐C6H3). Infinite chain structures of the parent sodium and potassium amides are disclosed, adding to the few known crystallographically characterised unsolvated s‐block metal amides. Solvation by N,N,N′,N′′,N′′‐pentamethyldiethylenetriamine (PMDETA) or N,N,N′,N′‐tetramethylethylenediamine (TMEDA) gives molecular variants of the lithium and sodium amides; whereas for potassium, PMDETA gives a molecular structure, TMEDA affords a novel, hemi‐solvated infinite chain. Crystal structures of the first magnesiate examples of this amide in [MMg{N(SiMe3)(Dipp)}2(μ‐nBu)]∞ (M=Na or K) are also revealed, though these breakdown to their homometallic components in donor solvents as revealed through NMR and DOSY studies. PMID:27573676

  16. Phase II clinical trial using californium 252 fast neutron brachytherapy, external pelvic radiation, and extrafascial hysterectomy in the treatment of bulky, barrel-shaped stage IB cervical cancer.

    PubMed

    van Nagell, J R; Maruyama, Y; Donaldson, E S; Hanson, M B; Gallion, H H; Yoneda, J; Powell, D E; Kryscio, R J; Beach, J L

    1986-05-15

    From June 1977 to June 1983, 32 patients with bulky (greater than 4 cm diameter), barrel-shaped Stage IB cervical cancer were treated at the University of Kentucky Medical Center by a combination of outpatient neutron brachytherapy using californium 252 (252Cf) and external pelvic radiation followed by extrafascial hysterectomy. Nineteen patients had cervical tumors 4 to 6 cm in diameter, and 13 patients had lesions in excess of 6 cm in diameter. A dose of 4500 rad external photon therapy was given from a linear accelerator, and one or two 6-hour 252Cf implants were given during or immediately after external radiation. Extrafascial hysterectomy with bilateral salpingo-oophorectomy was performed 6 weeks after completion of radiation therapy. Complications during and after radiation were minimal and included vaginal stenosis (three) and proctitis (two). Tumor clearance in the hysterectomy specimen was complete in 23 patients (72%) and residual cervical tumor was present in 9 patients (28%). Two patients developed tumor recurrence and died of disease 15 and 27 months after therapy, respectively. Thirty patients remain free of disease 26 to 96 months (median, 52 months) after treatment, and none have been lost to follow-up. The actuarial survival of these patients is 97% at 2 years and 94% at 5 years. Intracavitary neutron therapy is well tolerated and is effective when combined with external radiation and hysterectomy in the treatment of bulky, barrel-shaped Stage IB cervical cancer.

  17. Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts.

    PubMed

    Diaz-Parga, Pedro; Goto, Joy J; Krishnan, V V

    2017-09-18

    Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.

  18. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    PubMed

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin

    2014-04-21

    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

  19. Quantification of DNA adducts in individual cells by immunofluorescence: effects of variation in DNA conformation.

    PubMed

    Frank, Adrian J; Tilby, Michael J

    2003-02-15

    Previously reported detection of melphalan-DNA adducts by immunofluorescent staining indicated considerable intercell variation in fluorescence levels. Investigations were undertaken to determine whether this variation reflected actual intercell differences in adduct levels. Melphalan-treated CCRF-CEM leukaemia cells were analysed by the trapped-in-agarose DNA immunostaining (TARDIS) method using fluorescein immunofluorescence and Hoechst dye-DNA fluorescence. Increasing the time of DNA denaturation in alkali affected the staining intensity, in agreement with known adduct properties, but failed to reduce intercell heterogeneity. To test the hypothesis that heterogeneity resulted from variation in levels of DNA strand breaks, drug-treated cells were exposed to ionising radiation. An increase in level and reduction in heterogeneity of immunofluorescence were observed, optimal at 10 Gy. When samples were irradiated after lysis, 1 Gy was optimal. At the optimal doses, irradiation before or after lysis resulted in similar levels of DNA strand breaks. Our conclusions are as follows: (a) There was no major intercell variation in the number of adducts other than from variation in DNA content. (b) Detection of melphalan, and possibly other adducts, by immunofluorescence can be markedly influenced by the level of strand breaks present in the DNA. (c) Samples analysed for melphalan adducts by immunofluorescence should be irradiated to minimise errors due to this factor. Copyright 2003 Elsevier Science (USA)

  20. Malondialdehyde–Deoxyguanosine Adducts among Workers of a Thai Industrial Estate and Nearby Residents

    PubMed Central

    Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Ceppi, Marcello; Sangrajrang, Suleeporn; Piro, Sara; Boffetta, Paolo

    2010-01-01

    Background Humans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. Objective We measured the levels of malondialdehyde–deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources. Design We conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution. Results The multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A nonsignificant increase in DNA adducts was observed after 4–6 years of employment among the petrochemical complexes. Conclusions Air pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution. PMID:20056580

  1. Reaction of epichlorohydrin with adenosine, 2'-deoxyadenosine and calf thymus DNA: identification of adducts.

    PubMed

    Sund, Pernilla; Kronberg, Leif

    2006-06-01

    Epichlorohydrin (a probable human carcinogen) was allowed to react with adenosine and the adducts were characterized by NMR and UV spectroscopy, and mass spectrometry. The adduct initially formed was 1-(3-chloro-2-hydroxypropyl)-adenosine, which subsequently ring closures to 1,N(6)-(2-hydroxypropyl)-adenosine at neutral and basic conditions. At acid conditions, the N-1 adduct undergoes a slow deamination to yield 1-(3-chloro-2-hydroxypropyl)-inosine. Minor adducts identified were 7-(3-chloro-2-hydroxypropyl)-adenosine and 3-(3-chloro-2-hydroxypropyl)-adenosine which are easily deglycosylated, and an adduct where the epichlorohydrin residue was attached to the sugar moiety of adenosine. A diadduct, 1,N(6)-(2-hydroxypropyl)-N(6)-(3-chloro-2-hydroxypropyl)-adenosine was also identified. The reaction of epichlorohydrin with calf thymus DNA gave 1,N(6)-(2-hydroxypropyl)-deoxyadenosine and 3-(3-chloro-2-hydroxypropyl)-adenine (major adduct).

  2. Effects of metal ion adduction on the gas-phase conformations of protein ions.

    PubMed

    Flick, Tawnya G; Merenbloom, Samuel I; Williams, Evan R

    2013-11-01

    Changes in protein ion conformation as a result of nonspecific adduction of metal ions to the protein during electrospray ionization (ESI) from aqueous solutions were investigated using traveling wave ion mobility spectrometry (TWIMS). For all proteins examined, protein cations (and in most cases anions) with nonspecific metal ion adducts are more compact than the fully protonated (or deprotonated) ions with the same charge state. Compaction of protein cations upon nonspecific metal ion binding is most significant for intermediate charge state ions, and there is a greater reduction in collisional cross section with increasing number of metal ion adducts and increasing ion valency, consistent with an electrostatic interaction between the ions and the protein. Protein cations with the greatest number of adducted metal ions are no more compact than the lowest protonated ions formed from aqueous solutions. These results show that smaller collisional cross sections for metal-attached protein ions are not a good indicator of a specific metal-protein interaction in solution because nonspecific metal ion adduction also results in smaller gaseous protein cation cross sections. In contrast, the collisional cross section of α-lactalbumin, which specifically binds one Ca(2+), is larger for the holo-form compared with the apo-form, in agreement with solution-phase measurements. Because compaction of protein cations occurs when metal ion adduction is nonspecific, elongation of a protein cation may be a more reliable indicator that a specific metal ion-protein interaction occurs in solution.

  3. Effects of Metal Ion Adduction on the Gas-Phase Conformations of Protein Ions

    PubMed Central

    Flick, Tawnya G.; Merenbloom, Samuel I.; Williams, Evan R.

    2013-01-01

    Changes in protein ion conformation as a result of nonspecific adduction of metal ions to the protein during electrospray ionization (ESI) from aqueous solutions were investigated using traveling wave ion mobility spectrometry (TWIMS). For all proteins examined, protein cations (and in most cases anions) with nonspecific metal ion adducts are more compact than the fully protonated (or deprotonated) ions with the same charge state. Compaction of protein cations upon nonspecific metal ion binding is most significant for intermediate charge state ions, and there is a greater reduction in collisional cross section with increasing number of metal ion adducts and increasing ion valency, consistent with an electrostatic interaction between the ions and the protein. Protein cations with the greatest number of adducted metal ions are no more compact than the lowest protonated ions formed from aqueous solutions. These results show that smaller collisional cross sections for metal-attached protein ions are not a good indicator of a specific metal-protein interaction in solution, because nonspecific metal ion adduction also results in smaller gaseous protein cation cross sections. In contrast, the collisional cross section of α-lactalbumin, which specifically binds one Ca2+, is larger for the holo-form compared to the apo-form, in agreement with solution-phase measurements. Because compaction of protein cations occurs when metal ion adduction is nonspecific, elongation of a protein cation may be a more reliable indicator that a specific metal ion-protein interaction occurs in solution. PMID:23733259

  4. In vitro characterization of DNA adducts formed by foundry air particulate matter.

    PubMed

    Savela, K; Kohan, M J; Walsh, D; Perera, F P; Hemminki, K; Lewtas, J

    1996-05-01

    This study is part of an ongoing investigation of biomarkers in iron foundry workers exposed to polycyclic aromatic compounds. Foundry workers with the highest exposures had elevated levels of DNA adducts in their white blood cells in previous studies. The purpose of this study was to characterize the nature of DNA reactive chemicals in foundry air samples through incubating the foundry filter extract with DNA and activation enzymes. Calf thymus DNA was incubated with foundry filter extract and activated by either rat liver activation mixture (S9 mix) or xanthine oxidase. A complex pattern of adducts was observed on thin-layer chromatography (TLC) by the 32P-postlabeling assay. Two selected polycyclic aromatic hydrocarbons (PAHs)--1-NP-and anti(+/-)benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide [anti(+/-) BPDE]-DNA adducts--were used as marker compounds in characterizing the postlabeled DNA adducts by TLC combined with high-performance liquid chromatography (HPLC). After an initial separation of DNA adducts by TLC, individual spots were isolated and separated further on HPLC. HPLC analysis and spiking with anti(+/-)BPDE-DNA standard confirmed the co-migration of the anti(+/-)BPDE-DNA standard with one PAH adduct formed by the S9 mix-activated DCM extract in calf thymus DNA.

  5. In vitro characterization of DNA adducts formed by foundry air particulate matter.

    PubMed Central

    Savela, K; Kohan, M J; Walsh, D; Perera, F P; Hemminki, K; Lewtas, J

    1996-01-01

    This study is part of an ongoing investigation of biomarkers in iron foundry workers exposed to polycyclic aromatic compounds. Foundry workers with the highest exposures had elevated levels of DNA adducts in their white blood cells in previous studies. The purpose of this study was to characterize the nature of DNA reactive chemicals in foundry air samples through incubating the foundry filter extract with DNA and activation enzymes. Calf thymus DNA was incubated with foundry filter extract and activated by either rat liver activation mixture (S9 mix) or xanthine oxidase. A complex pattern of adducts was observed on thin-layer chromatography (TLC) by the 32P-postlabeling assay. Two selected polycyclic aromatic hydrocarbons (PAHs)--1-NP-and anti(+/-)benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide [anti(+/-) BPDE]-DNA adducts--were used as marker compounds in characterizing the postlabeled DNA adducts by TLC combined with high-performance liquid chromatography (HPLC). After an initial separation of DNA adducts by TLC, individual spots were isolated and separated further on HPLC. HPLC analysis and spiking with anti(+/-)BPDE-DNA standard confirmed the co-migration of the anti(+/-)BPDE-DNA standard with one PAH adduct formed by the S9 mix-activated DCM extract in calf thymus DNA. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 1. E Figure 1. F PMID:8781406

  6. Formation of dopamine adducts derived from brain polyunsaturated fatty acids: mechanism for Parkinson disease.

    PubMed

    Liu, Xuebo; Yamada, Naruomi; Maruyama, Wakako; Osawa, Toshihiko

    2008-12-12

    Oxidative stress appears to be directly involved in the pathogenesis of the neurodegeneration of dopaminergic systems in Parkinson disease. In this study, we formed four dopamine modification adducts derived from docosahexaenoic acid (C22:6/omega-3) and arachidonic acid (C18:4/omega-6), which are known as the major polyunsaturated fatty acids in the brain. Upon incubation of dopamine with fatty acid hydroperoxides and an in vivo experiment using rat brain tissue, all four dopamine adducts were detected. Furthermore, hexanoyl dopamine (HED), an arachidonic acid-derived adduct, caused severe cytotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells, whereas the other adducts were only slightly affected. The HED-induced cell death was found to include apoptosis, which also seems to be mediated by reactive oxygen species generation and mitochondrial abnormality. Additionally, the experiments using monoamine transporter inhibitor and mouse embryonic fibroblast NIH-3T3 cells that lack the monoamine transporter indicate that the HED-induced cytotoxicity might specially occur in the neuronal cells. These data suggest that the formation of the docosahexaenoic acid- and arachidonic acid-derived dopamine adducts in vitro and in vivo, and HED, the arachidonic acid-derived dopamine modification adduct, which caused selective cytotoxicity of neuronal cells, may indicate a novel mechanism responsible for the pathogenesis in Parkinson disease.

  7. Single Molecule Study on Incorporation Efficiency of DPO4 and Klenow Fragment to BPDE Adduct

    NASA Astrophysics Data System (ADS)

    Song, Lu; Yeh, Yin; Balhorn, Rod; Cosman, Monique

    2009-03-01

    DNA synthesis involving high fidelity A-family polymerases such as Klenow fragment is blocked by DNA adducts, while Y-family DNA polymerases such as Dpo4 can bypass the DNA adducts to resume DNA synthesis. So understanding the functional relationship between A-family and Y-family DNA polymerases in DNA replication and the mechanism of bypassing DNA adducts is of great help to explain the cause of mutagenesis. We introduce a flow cell on modified surface to study the incorporation efficiency of Dpo4 and Klenow fragments to benzo[a]pyrene-diol-epoxide (BPDE) adduct at single molecule level. Specifically, we anchor the labeled DNA onto the modified surface with adduct site open for nucleotide incorporation and flow the polymerases and labeled nucleotides into flow cell. With Total Internal Reflection Fluorescence Microscopy (TIRFM) we identify the incorporation of the nucleotides onto the anchored DNA template by identifying the co-localization of the template position and that of the labeled nucleotide. We further quantify the signal densities of the images obtained from the two different polymerases, thus examining whether incorporation reactions have been executed and quantifying the incorporation efficiency of the polymerases. We can also identify, on the specific adduct site, which nucleotide, if any, is incorporated by each of the two polymerases.

  8. Malondialdehyde-deoxyguanosine adducts among workers of a Thai industrial estate and nearby residents.

    PubMed

    Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Ceppi, Marcello; Sangrajrang, Suleeporn; Piro, Sara; Boffetta, Paolo

    2010-01-01

    Humans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. We measured the levels of malondialdehyde-deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources. We conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution. The multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A nonsignificant increase in DNA adducts was observed after 4-6 years of employment among the petrochemical complexes. Air pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution.

  9. Assay of Protein and Peptide Adducts of Cholesterol Ozonolysis Products by Hydrophobic and Click Enrichment Methods

    PubMed Central

    2015-01-01

    Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods—Folch extraction and solid phase extraction (SPE)—were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide–seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and asthma. PMID:25185119

  10. Assay of protein and peptide adducts of cholesterol ozonolysis products by hydrophobic and click enrichment methods.

    PubMed

    Windsor, Katherine; Genaro-Mattos, Thiago C; Miyamoto, Sayuri; Stec, Donald F; Kim, Hye-Young H; Tallman, Keri A; Porter, Ned A

    2014-10-20

    Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods-Folch extraction and solid phase extraction (SPE)-were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide-seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, and asthma.

  11. Seasonal and intertidal impact on DNA adduct levels in gills of blue mussels (Mytilus edulis L.).

    PubMed

    Skarphédinsdóttir, Halldóra; Ericson, Gunilla; Halldórsson, Halldór P; Svavarsson, Jörundur

    2005-07-01

    The aim of this study was to elucidate possible seasonal variation in DNA adduct levels in blue mussels (Mytilus edulis), and to investigate the impact of intertidal exposure on the DNA adduct levels, i.e. to explore if DNA adduct levels in mussels in the intertidal zone differ from those in the subtidal zone. Blue mussels were deployed separately in the intertidal and subtidal zone at a contaminated and a reference site in Iceland, and sampled regularly during one year. Gill DNA adduct levels were found to be higher in mussels in the intertidal zone compared to the subtidal zone at the contaminated site, the difference being largest in winter. Total PAH tissue levels were also higher in mussels in the intertidal zone. Seasonal variation was observed in both DNA adduct and PAH tissue levels in mussels at the contaminated site, with lower levels from the time of transplantation in summer to autumn, maximum levels in winter, which decreased to lower levels again in spring and summer the following year. DNA adducts and PAH levels were low or below the detection limits in mussels at the reference site at all times, both in the intertidal and subtidal zone.

  12. Cigarette smoke-induced DNA adducts in the respiratory and nonrespiratory tissues of rats

    SciTech Connect

    Gairola, C.G.; Gupta, R.C. )

    1991-01-01

    Formation of DNA adducts is regarded as an essential initial step in the process of chemical carcinogenesis. To determine how chronic exposure to cigarette smoke affects the distribution of DNA adducts in selected respiratory and nonrespiratory tissues. The authors exposed male Sprague-Dawley rats daily to fresh mainstream smoke from the Univ. of Kentucky reference cigarettes (2R1) in a nose-only exposure system for 32 weeks. Blood carboxyhemoglobin, total particulate matter (TPM) intake, and pulmonary aryl hydrocarbon hydroxylase values indicated effective exposure of animals to cigarette smoke. DNA was extracted from three respiratory (larynx, trachea, and lung) and three nonrespiratory (liver, heart, and bladder) tissues and analyzed for DNA adducts by the {sup 32}P-postlabeling assay under conditions capable of detecting low levels of diverse aromatic/hydrophobic adducts. Data showed that the total DNA adducts in the lung, heart, and trachea, and larynx were increased by 10- to 20-fold in the smoke-exposed group. These data suggest selective formation of DNA adducts in the tissues.

  13. Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

    PubMed Central

    2016-01-01

    Serum albumin (Alb) is the most abundant protein in blood plasma. Alb reacts with many carcinogens and/or their electrophilic metabolites. Studies conducted over 20 years ago showed that Alb forms adducts with the human carcinogens aflatoxin B1 and benzene, which were successfully used as biomarkers in molecular epidemiology studies designed to address the role of these chemicals in cancer risk. Alb forms adducts with many therapeutic drugs or their reactive metabolites such as β-lactam antibiotics, acetylsalicylic acid, acetaminophen, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, and antiretroviral therapy drugs. The identification and characterization of the adduct structures formed with Alb have served to understand the generation of reactive metabolites and to predict idiosyncratic drug reactions and toxicities. The reaction of candidate drugs with Alb is now exploited as part of the battery of screening tools to assess the potential toxicities of drugs. The use of gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry (LC-MS) enabled the identification and quantification of multiple types of Alb xenobiotic adducts in animals and humans during the past three decades. In this perspective, we highlight the history of Alb as a target protein for adduction to environmental and dietary genotoxicants, pesticides, and herbicides, common classes of medicinal drugs, and endogenous electrophiles, and the emerging analytical mass spectrometry technologies to identify Alb-toxicant adducts in humans. PMID:27989119

  14. Acetaminophen protein adduct formation following low-dose acetaminophen exposure: comparison of immediate-release vs extended-release formulations.

    PubMed

    James, Laura P; Chiew, Angela; Abdel-Rahman, Susan M; Letzig, Lynda; Graudins, Andis; Day, Peter; Roberts, Dean

    2013-04-01

    Acetaminophen (APAP) protein adducts are a biomarker of APAP metabolism, reflecting oxidation of APAP and generation of the reactive metabolite N-acetyl-p-benzoquinone imine. High levels of adducts correspond to liver toxicity in patients with APAP-related acute liver failure. Adduct formation following low-dose exposure to APAP has not been well studied. APAP protein adducts were measured in blood samples collected from fasted individuals who participated in a crossover study of APAP (80 mg/kg) comparing extended release (ER) and immediate release (IR) formulations. Adducts were quantified in all postdose blood samples using a validated high-performance liquid chromatography electrochemical detection (HPLC-EC) assay. Comparison of pharmacokinetic parameters for adducts did not reveal significant differences between ER and IR formulations, with one exception. Formation rates for adducts were faster for IR than the ER formulation (0.420 ± 0.157 vs. 0.203 ± 0.080 1/h), respectively. Maximum plasma concentrations (Cmax) of adducts for IR and ER were 0.108 (±0.020) and 0.100 (±0.028) nmol/ml serum, respectively, and were two orders of magnitude lower than adduct levels previously reported in adults with acute liver failure secondary to APAP. APAP protein adducts are rapidly formed following nontoxic ingestion of APAP at levels significantly lower than those associated with acute liver failure.

  15. The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts

    PubMed Central

    Wyss, Laura A.; Nilforoushan, Arman; Williams, David M.; Marx, Andreas; Sturla, Shana J.

    2016-01-01

    Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O6-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O6-methylguanine (O6-MeG) and O6-carboxymethylguanine (O6-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2′-deoxynucleoside-5′-O-triphosphates (BenziTP and BIMTP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide Benzi opposite adducts, with up to 150-fold higher catalytic efficiency for O6-MeG over guanine in the template. Furthermore, addition of artificial nucleotide Benzi was required for full-length DNA synthesis during bypass of O6-CMG. Selective incorporation of the artificial nucleotide opposite an O6-alkylguanine DNA adduct was verified using a novel 2′,3′-dideoxy derivative of BenziTP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BenziTP opposite biologically relevant O6-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies. PMID:27378785

  16. Acetaminophen protein adduct formation following low dose acetaminophen exposure: comparison of immediate release vs. extended release formulations

    PubMed Central

    James, Laura P.; Chiew, Angela; Abdel-Rahman, Susan M.; Letzig, Lynda; Graudins, Andis; Day, Peter; Roberts, Dean

    2012-01-01

    Purpose Acetaminophen (APAP) protein adducts are a biomarker of APAP metabolism, reflecting oxidation of APAP and generation of the reactive metabolite N-acetyl-p-benzoquinone imine. High levels of adducts correspond to liver toxicity in patients with APAP related acute liver failure. Adduct formation following low dose exposure to APAP has not been well studied. APAP protein adducts were measured in blood samples collected from fasted subjects that participated in a cross-over study of APAP (80 mg/kg) comparing extended release (ER) and immediate release (IR) formulations. Methods Adducts were quantified in all post-dose blood samples using a validated HPLC-EC assay. Results Comparison of pharmacokinetic parameters for adducts did not reveal significant differences between the ER and IR formulations, with one exception. Formation rates for adducts were faster for the IR than the ER formulation (0.420 ± 0.157 vs. 0.203 ± 0.080 1/hr), respectively. The Cmax of adducts for IR and ER were 0.108 (±0.020) and 0.100 (±0.028) nmol/mL serum, respectively, and were two orders of magnitude lower than adduct levels previously reported in adults with acute liver failure secondary to APAP. Conclusions APAP protein adducts are rapidly formed following non-toxic ingestion of APAP at levels significantly lower than those associated with acute liver failure. PMID:23052410

  17. Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro.

    PubMed

    Reddy, M V; Blackburn, G R; Schreiner, C A; Mackerer, C R

    1997-08-01

    An in vitro system was utilized to measure DNA adduct-forming ability of petroleum oils and oil coal tar mixtures to define correlations between DNA adduct levels and their mutagenic potencies. The system consisted of reaction of dimethyl sulfoxide extracts of oils with calf thymus DNA in the presence of Aroclor-induced hamster liver microsomes for 30 min. Following DNA extraction, DNA adducts were measured by the nuclease P1-enhanced postlabeling assay coupled with two-dimensional polyethyleneimine (PEI)-cellulose TLC. Thin layer plates showed putative aromatic DNA adducts, with levels ranging from 60 to 1400 adducts per 10(9) DNA nucleotides. TLC mobilities suggested adducts to be aromatic compounds containing 4 or more rings. A good correlation (coefficient of correlation = 0.91) was observed between DNA adduct levels and Salmonella mutagenicity for 19 oils. All 19 samples tested produced DNA adducts. To expedite the TLC procedure, adducts were resolved by one-dimensional TLC and the radioactivity measured using a mechanical scanner. Results were comparable to those obtained by two-dimensional TLC and quantification after scraping. Our data show that the in vitro incubation system coupled with the postlabeling adduct assay is a useful screening method to identify mutagenic and potentially carcinogenic oils.

  18. Identification of Rosmarinic Acid-Adducted Sites in Meat Proteins in a Gel Model under Oxidative Stress by Triple TOF MS/MS.

    PubMed

    Tang, Chang-Bo; Zhang, Wan-Gang; Wang, Yao-Song; Xing, Lu-Juan; Xu, Xing-Lian; Zhou, Guang-Hong

    2016-08-24

    Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation.

  19. Persistence of benzo[a]pyrene--DNA adducts in hematopoietic tissues and blood of the mummichog, Fundulus heteroclitus.

    PubMed

    Rose, W L; French, B L; Reichert, W L; Faisal, M

    2001-05-01

    The formation and persistence of benzo[a]pyrene (B[a]P)-DNA adducts were investigated in blood, liver and two hematopoietic tissues (anterior kidney and spleen) of the mummichog (Fundulus heteroclitus). Fish were injected with a single, sublethal dose of B[a]P (12 mg/kg body weight) and sampled from 8 to 96 days post-injection. 32P-Postlabeling analysis and storage phosphor imaging were used to resolve and quantify hydrophobic DNA adducts. One major DNA adduct was present in each of the examined tissues at all sampling times. This adduct had similar chromatographic characteristics to those of the adduct standard, 7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydro-benzo[a]pyrene (B[a]PDE-dG). Minor DNA adduct spots, representing less than 2% of the total DNA adducts, were observed in some liver, anterior kidney and spleen samples for up to 32 days post-injection. The B[a]P-DNA adducts reached maximal levels at 32 days post-injection and persisted for at least 96 days in all examined tissues. B[a]P-DNA adduct levels were significantly higher in the liver and anterior kidney than in the spleen from 16 to 96 days (P<0.001), although liver and anterior kidney DNA adduct levels were not significantly different at any time. This is the first controlled study to demonstrate the formation and persistence of B[a]P-DNA adducts in hematopoietic tissues and blood of fishes exposed to the prototypical polycyclic aromatic hydrocarbon, B[a]P. Although persistent DNA adducts are generally recognized as potential initiators of carcinogenic processes, adducts in these vital tissues may also lead to disruption of physiological functions such defense mechanisms and hematopoiesis.

  20. Benzo(a)pyrene-albumin