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Sample records for 4-hydroxynonenal adduct accumulation

  1. Increased accumulation of 4-hydroxynonenal adducts in male GSTA4/PPAR alpha double knockout mice enhances injury during early stages of alcoholic liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatic lipid peroxidation and accumulation of aldehyde-adducted proteins occur early in alcohol-mediated injury and are postulated to mediate the subsequent pro-inflammatory and fibrotic responses observed in alcoholic liver disease. To test the significance of lipid peroxidation formation in the ...

  2. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload.

    PubMed Central

    Houglum, K; Filip, M; Witztum, J L; Chojkier, M

    1990-01-01

    In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investigated whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA- and 4-HNE- protein adducts. Chronic iron feeding resulted in hepatic iron overload (greater than 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDA- and 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a approximately 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized. Images PMID:2123889

  3. Protein adducts of malondialdehyde and 4-hydroxynonenal in livers of iron loaded rats: quantitation and localization.

    PubMed

    Khan, M Firoze; Wu, Xiaohong; Tipnis, Ulka R; Ansari, G A S; Boor, Paul J

    2002-05-01

    Pathophysiological mechanisms for hepatocellular injury, fibrosis and/or cirrhosis in hepatic iron overload are poorly understood. An increase in intracellular transit pool of iron can catalyze peroxidation of lipids to produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Covalent binding of such lipid aldehydes with proteins may cause impairment in cellular function and integrity. This investigation was focused on quantitative determination of MDA and HNE-protein adducts, and to establish a correlation between iron deposition and formation and localization of MDA and HNE-protein adducts, using immunohistochemistry. To achieve iron overload, male SD rats were fed a 2.5% carbonyl iron-supplemented diet for six weeks, while control animals received standard diet. Total iron as well as low molecular weight chelatable iron (LMWC-Fe) in the hepatic tissue of rats fed the iron supplemented diet increased significantly ( approximately 14- and approximately 15-fold, respectively). Quantitative ELISA for MDA-and HNE-protein adducts showed remarkable increases of 186 and 149%, respectively, in the liver homogenates of rats fed the iron-supplemented diet. Sections of liver stained for iron showed striking iron deposits in periportal (zone 1) hepatocytes, which was less dramatic in midzonal (zone 2) cells. Livers from iron-loaded rats showed strong, diffuse staining for both MDA and HNE adducts, which was highly pronounced in centrilobular (zone 3) hepatocytes, but was also evident in midzonal cells (zone 2). The demonstration of greater formation of both MDA and HNE-protein adducts provides evidence of iron-catalyzed lipid peroxidation in vivo. Although in this model of iron overload there was no evidence of tissue injury, our results provide an account of some of the initiating factors or early molecular events in hepatocellular damage that may lead to the pathological manifestations seen in chronic iron overload.

  4. Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro.

    PubMed

    Jørgensen, Peter; Milkovic, Lidija; Zarkovic, Neven; Waeg, Georg; Rattan, Suresh I S

    2014-02-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

  5. S-adenosyl-L-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry

    SciTech Connect

    Brown, James Mike; Kuhlman, Christopher; Terneus, Marcus V.; Labenski, Matthew T.; Lamyaithong, Andre Benja; Ball, John G.; Lau, Serrine S.; Valentovic, Monica A.

    2014-12-01

    Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-L-methionine (SAMe) treatment 1 hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n = 5/group) were divided into the following groups and treated as indicated: Veh (15 ml/kg water, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg), and SAMe given 1 h after APAP (S + A). APAP toxicity was confirmed by an increase (p < 0.05) in plasma ALT (U/l) and liver weight/10 g body weight relative to the Veh, SAMe and S + A groups 4 h following APAP treatment. SAMe administered 1 h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10 g body weights were lower in the S + A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S + A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1. - Highlights: • Acetaminophen (APAP) toxicity protected by S-adenosylmethionine (SAMe) • 4-Hydroxynonenal adducted to sarcosine dehydrogenase • 4-Hydroxynonenal adducted to carbamoyl phosphate synthetase-1 • SAMe reduced APAP mediated CPS-1 mitochondrial leakage.

  6. 4-hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake.

    PubMed

    Springer, J E; Azbill, R D; Mark, R J; Begley, J G; Waeg, G; Mattson, M P

    1997-06-01

    Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4-hydroxynonenal (HNE), which cross-links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein-bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein-bound HNE increased in content in the damaged spinal cord at early times following injury (1-24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein-bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose-dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms. PMID:9166741

  7. Conformational Interconversion of the trans-4-Hydroxynonenal-Derived (6S,8R,11S) 1,N2-Deoxyguanosine Adduct When Mismatched with Deoxyadenosine in DNA

    PubMed Central

    Huang, Hai; Wang, Hao; Lloyd, R. Stephen; Rizzo, Carmelo J.; Stone, Michael P.

    2009-01-01

    The (6S,8R,11S) 1,N2-HNE-dG adduct of trans-4-hydroxynonenal (HNE) was incorporated into the duplex 5′-d(GCTAGCXAGTCC)-3′•5′-d(GGACTAGCTAGC)-3′ [X=(6S,8R,11S) HNE-dG], in which the lesion was mismatched opposite dA. The (6S,8R,11S) adduct maintained the ring-closed 1,N2-HNE-dG structure. This was in contrast to when this adduct was correctly paired with dC, conditions under which it underwent ring opening and re-arrangement to diastereomeric minor groove hemiacetals [Huang, H., Wang, H., Qi, N., Lloyd, R.S., Harris, T.M., Rizzo, C.J., & Stone, M.P. (2008) J. Am. Chem. Soc. 130, 10898–10906]. The (6S,8R,11S) adduct exhibited a syn/anti conformational equilibrium about the glycosyl bond. The syn conformation was predominant in acidic solution. Structural analysis of the syn conformation revealed that X7 formed a distorted base pair with the complementary protonated A18. The HNE moiety was located in the major groove. Structural perturbations were observed at the neighbor C6•G19 and A8•T17 base pairs. At basic pH, the anti conformation of X7 was the major species. At X7 the 1,N2-HNE-dG intercalated and displaced the complementary A18 in the 5′-direction, resulting in a bulge at the X7•A18 base pair. The HNE aliphatic chain was oriented towards the minor groove. The Watson-Crick hydrogen bonding of the neighboring A8•T17 base pair was also disrupted. PMID:19053179

  8. Fat accumulation in Caenorhabditis elegans triggered by the electrophilic lipid peroxidation product 4-Hydroxynonenal (4-HNE)

    PubMed Central

    Singh, Sharda P.; Niemczyk, Maciej; Zimniak, Ludwika; Zimniak, Piotr

    2009-01-01

    Deposition and mobilization of fat in an organism are tightly controlled by multiple levels of endocrine and neuroendocrine regulation. Because these hormonal mechanisms ultimately act by affecting biochemical reactions of fat synthesis or utilization, obesity could be also modulated by altering directly the underlying lipid biochemistry. We have previously shown that genetically modified mice with an elevated level of the lipid peroxidation product 4-HNE become obese. We now demonstrate that the process is phylogenetically conserved and thus likely to be universal. In the nematode C. elegans, disruption of either conjugation or oxidation of 4-HNE leads to fat accumulation, whereas augmentation of 4-HNE conjugation results in a lean phenotype. Moreover, direct treatment of C. elegans with synthetic 4-HNE causes increased lipid storage, directly demonstrating a causative role of 4-HNE. The postulated mechanism, which involves modulation of acetyl-CoA carboxylase activity, could contribute to the triggering and maintenance of the obese phenotype on a purely metabolic level. PMID:20157589

  9. Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal

    PubMed Central

    2009-01-01

    The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses

  10. Elevated oxidative stress, iron accumulation around microvessels and increased 4-hydroxynonenal immunostaining in zone 1 of the liver acinus in hypercholesterolemic rabbits.

    PubMed

    Ong, Wei-Yi; Jenner, Andrew M; Pan, Ning; Ong, Choon-Nam; Halliwell, Barry

    2009-03-01

    Rabbits were fed a diet containing 1% cholesterol for 8 weeks and the levels of iron and oxidized lipids in liver analysed using atomic absorption spectroscopy and gas chromatography-mass spectrometry. A non-significant trend to an increase in iron level, but significant increases in the lipid peroxidation products, F(2)-isoprostanes and the cholesterol oxidation products 7 beta hydroxycholesterol, 7 ketocholesterol and cholesterol 5,6-alpha epoxide were detected in the liver of the cholesterol-fed rabbits. Histological analysis showed greater accumulation of lipids by Sudan red labelling in hepatocytes of zone I than zones II and III of the liver acinus. The increase in lipids coincided with an increase in iron staining in macrophages around liver microvessels and increased immunostaining to melanotransferrin and the lipid peroxidation product, 4-hydroxynonenal (4-HNE), in zone 1. The results are suggestive of microvascular damage associated with iron accumulation and oxidative stress in the liver during hypercholesterolemia.

  11. Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N[superscript 2]-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV

    SciTech Connect

    Banerjee, Surajit; Christov, Plamen P.; Kozekova, Albena; Rizzo, Carmelo J.; Egli, Martin; Stone, Michael P.

    2014-10-02

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of {omega}-6 polyunsaturated fatty acids in vivo. Michael addition of the N{sub 2}-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N{sub 2}-dGuo (1,N{sub 2}-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua {yields} Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, the (6S,8R,11S)-1,N{sub 2}-dGuo lesion remained in the ring

  12. Malondialdehyde and 4-hydroxynonenal adducts are not formed on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in diabetes.

    PubMed

    Moore, Caronda J; Shao, Chun Hong; Nagai, Ryoji; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R

    2013-04-01

    Recently, we reported an elevated level of glucose-generated carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca(2+) transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes? This study used western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, N (ε)-carboxy(methyl)lysine (CML), pentosidine, and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca(2+) kinetics in myocytes from control, diabetic, and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from either control or 8 weeks diabetic rats with altered evoked Ca(2+) transients. However, CML, pentosidine, and pyrraline adducts were elevated three- to five-fold (p < 0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-RCS scavenger) reduced CML, pentosidine, and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca(2+) cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca(2+) cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca(2+) dysregulation.

  13. Differential metabolism of 4-hydroxynonenal in liver, lung and brain of mice and rats

    SciTech Connect

    Zheng, Ruijin; Dragomir, Ana-Cristina; Mishin, Vladimir; Richardson, Jason R.; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-08-15

    The lipid peroxidation end-product 4-hydroxynonenal (4-HNE) is generated in tissues during oxidative stress. As a reactive aldehyde, it forms Michael adducts with nucleophiles, a process that disrupts cellular functioning. Liver, lung and brain are highly sensitive to xenobiotic-induced oxidative stress and readily generate 4-HNE. In the present studies, we compared 4-HNE metabolism in these tissues, a process that protects against tissue injury. 4-HNE was degraded slowly in total homogenates and S9 fractions of mouse liver, lung and brain. In liver, but not lung or brain, NAD(P)+ and NAD(P)H markedly stimulated 4-HNE metabolism. Similar results were observed in rat S9 fractions from these tissues. In liver, lung and brain S9 fractions, 4-HNE formed protein adducts. When NADH was used to stimulate 4-HNE metabolism, the formation of protein adducts was suppressed in liver, but not lung or brain. In both mouse and rat tissues, 4-HNE was also metabolized by glutathione S-transferases. The greatest activity was noted in livers of mice and in lungs of rats; relatively low glutathione S-transferase activity was detected in brain. In mouse hepatocytes, 4-HNE was rapidly taken up and metabolized. Simultaneously, 4-HNE-protein adducts were formed, suggesting that 4-HNE metabolism in intact cells does not prevent protein modifications. These data demonstrate that, in contrast to liver, lung and brain have a limited capacity to metabolize 4-HNE. The persistence of 4-HNE in these tissues may increase the likelihood of tissue injury during oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a highly reactive aldehyde. • Rodent liver, but not lung or brain, is efficient in degrading 4-hydroxynonenal. • 4-hydroxynonenal persists in tissues with low metabolism, causing tissue damage.

  14. Increased accumulation of 4-hydroxynonenal adducts in female GSTA4/PPAR alpha double knockout mice enhance steatosis and inflammation in a model of pediatric nonalcoholic fatty liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatocellular injury resulting from increased lipid peroxidation products and oxidative stress is considered a potential mechanism driving the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitsis (NASH). To test the significance of lipid peroxidation and protein...

  15. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

    PubMed

    Kapphahn, Rebecca J; Giwa, Babatomiwa M; Berg, Kristin M; Roehrich, Heidi; Feng, Xiao; Olsen, Timothy W; Ferrington, Deborah A

    2006-07-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification. PMID:16530755

  16. Selective cleavage of thioether linkage in proteins modified with 4-hydroxynonenal.

    PubMed Central

    Uchida, K; Stadtman, E R

    1992-01-01

    The peroxidation of polyunsaturated fatty acids leads to numerous products, including 4-hydroxynonenal (HNE). That 4-hydroxy-2-alkenal compounds react with sulfhydryl groups of proteins to form thioether adducts possessing a carbonyl function has been established [Schauenstein, E. & Esterbauer, H. (1979) Ciba Found. Symp. 67, 225-244]. Taking advantage of the fact that Raney nickel catalyzes cleavage of thioether bonds, we have developed a procedure to quantitate the amount of HNE moiety bound to protein by means of a thioether linkage. Adducts of HNE with N-acetylcysteine and glutathione were prepared, labeled with NaB[3H]H4, and then treated with Raney nickel. The 3H-labeled product was recovered in 85-90% yield from both HNE-N-acetylcysteine and HNE-glutathione adducts in a solvent [10% (vol/vol) methanol/chloroform]-estractable form. Treatment of proteins with HNE led to the disappearance of protein sulfhydryl groups. However, less than 10% of the labeled adducts obtained after subsequent reduction with NaB[3H]H4 could be released in a solvent-extractable form upon treatment with Raney nickel. This and the observation that HNE reacts with proteins lacking a sulfhydryl group attests to the fact that HNE can react with amino acid residues other than cysteinyl residues. Images PMID:1608970

  17. 4-hydroxynonenal contributes to macrophage foam cell formation through increased expression of class A scavenger receptor at the level of translation.

    PubMed

    Yun, Mi R; Im, Dong S; Lee, Seung J; Woo, Joong W; Hong, Ki W; Bae, Sun S; Kim, Chi D

    2008-07-15

    4-Hydroxynonenal (HNE) is known to be atherogenic, but its mechanism of action in atherogenesis is not clear. Therefore, this study investigated the role of HNE in macrophage foam cell formation and the underlying mechanism involved in HNE-induced expression of scavenger receptors (SRs). In the aortic sinus of ApoE-deficient mice fed a high-fat diet, multiple plaque lesions were accompanied by increased accumulation of HNE adducts in the enhanced Mac-2 stained area. In an in vitro study, HNE exposure to J774A.1 macrophages led to increased expression of class A SR (SR-A) and CD36 at the protein level with a concomitant increase in endocytic uptake of oxLDL. In contrast to CD36 protein expression, which was associated with an increase in mRNA expression, the HNE-enhanced SR-A protein expression was neither accompanied by its mRNA expression nor affected by actinomycin D. HNE enhanced the incorporation rates of (35)S-Met/Cys into SR-A, and HNE-induced SR-A protein expression was effectively attenuated by translation inhibitors such as cycloheximide and rapamycin. Taken together, these data suggest that HNE contributes to macrophage foam cell formation through increased synthesis of SR-A at the level of mRNA translation, consequently leading to the progression of atherosclerosis.

  18. The oxidant role of 4-hydroxynonenal in corneal epithelium.

    PubMed

    Chen, Longlong; Zong, Rongrong; Zhou, Jing; Ge, Lianping; Zhou, Tong; Ma, Jian-xing; Liu, Zuguo; Zhou, Yueping

    2015-01-01

    4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in tissues and is reported to play pathogenic roles in eye diseases. Here we investigated the association between 4-HNE and oxidative stress in the corneal epithelium. 4-HNE suppressed the cell viability of human corneal epithelial cells (HCE) in a concentration dependent manner. 4-HNE significantly increased the level of 3-Nitrotyrosine (3-NT), a marker of oxidative stress, in HCE cells and corneal epithelium of rats by immunofluorescent staining and Western blot analysis. To its underlying mechanistic on ROS system, 4-HNE elevated the ROS generation enzyme NADPH oxidase 4 (NOX4) and induced the activation of NF-E2-related factor-2 (NRF2) and its downstream effectors: NAD(P)H dehydrogenase (quinone 1) (NQO1) and glutathione S-transferase P (GSTP). Furthermore, N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the inhibitory and oxidant effects of 4-HNE on the corneal epithelial cells. In conclusion, 4-HNE plays an oxidant role in the corneal epithelium and this work provides a new strategy for the pathogenesis and treatment of corneal diseases.

  19. Stereoselective Effects of 4-Hydroxynonenal in Cultured Mouse Hepatocytes

    PubMed Central

    Dabrowski, Michael J.; Zolnerciks, Joseph K.; Balogh, Larissa M.; Greene, Robert J.; Kavanagh, Terrance J.; Atkins, William M.

    2010-01-01

    4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 μM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. caspase-3-dependent cleavage of glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 hrs. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 hours after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms. PMID:20873854

  20. Role of 4-hydroxynonenal in chemopreventive activities of sulforaphane

    PubMed Central

    Sharma, Rajendra; Sharma, Abha; Chaudhary, Pankaj; Sahu, Mukesh; Jaiswal, Shailesh; Awasthi, Sanjay; Awasthi, Yogesh C.

    2012-01-01

    Chemoprevention of cancer via herbal and dietary supplements is a logical approach to combat cancer and presently it is an attractive area of research investigations. Over the years, the use of isothiocyanates, such as sulforaphane (SFN) found in cruciferous vegetables, has been advocated as chemopreventive agents and their efficacy has been demonstrated in cell lines and animal models. In-vivo studies with SFN suggest that besides protecting normal healthy cells from environmental carcinogens it also exhibits cytotoxicity and apoptotic effects against various cancer cell types. Among several mechanisms for the chemopreventive activity of SFN against chemical carcinogenesis, its effect on drug metabolizing enzymes that causes activation/ neutralization of carcinogenic metabolites is well established. Recent studies suggest that SFN exerts its selective cytotoxicity to cancer cells via reactive oxygen species (ROS)-mediated generation of lipid peroxidation (LPO) products particularly 4-hydroxynonenal (HNE). Against the background of the known biochemical effects of SFN on normal and cancer cells, in this article we have reviewed the underlying molecular mechanisms responsible for the overall chemopreventive effects of SFN focusing on the role of HNE in these mechanisms that may also contribute to its selective cytotoxicity to cancer cells. PMID:22579574

  1. Effects of 4-hydroxynonenal on vascular endothelial and smooth muscle cell redox signaling and function in health and disease☆

    PubMed Central

    Chapple, Sarah J.; Cheng, Xinghua; Mann, Giovanni E.

    2013-01-01

    4-hydroxynonenal (HNE) is a lipid hydroperoxide end product formed from the oxidation of n-6 polyunsaturated fatty acids. The relative abundance of HNE within the vasculature is dependent not only on the rate of lipid peroxidation and HNE synthesis but also on the removal of HNE adducts by phase II metabolic pathways such as glutathione-S-transferases. Depending on its relative concentration, HNE can induce a range of hormetic effects in vascular endothelial and smooth muscle cells, including kinase activation, proliferation, induction of phase II enzymes and in high doses inactivation of enzymatic processes and apoptosis. HNE also plays an important role in the pathogenesis of vascular diseases such as atherosclerosis, diabetes, neurodegenerative disorders and in utero diseases such as pre-eclampsia. This review examines the known production, metabolism and consequences of HNE synthesis within vascular endothelial and smooth muscle cells, highlighting alterations in mitochondrial and endoplasmic reticulum function and their association with various vascular pathologies. PMID:24024167

  2. Defining the mechanisms by which the reactive oxygen species by-product, 4-hydroxynonenal, affects human sperm cell function.

    PubMed

    Baker, Mark A; Weinberg, Anita; Hetherington, Louise; Villaverde, Ana-Izabel; Velkov, Tony; Baell, Jonathan; Gordon, Christopher P

    2015-04-01

    Lipid peroxidation products such as the naturally occurring aldehyde 4-hydroxynonenal (4-HNE) are known to be cytotoxic toward different cell types, including spermatozoa. In order to understand this at the molecular level, we have employed a proteomic approach to characterize direct 4-HNE adducts on human spermatozoa. Several proteins were identified to be of particular interest, including aldehyde labeling of histone methyltransferase and dynein heavy chain. In addition, we found that 4-HNE bound to part of the activation segment, cysteine residue 199, of protein kinase A (PKA). Interestingly, at low levels, addition of 4-HNE had a stimulatory effect on PKA. However, this did not correlate to increased phosphotyrosine levels during capacitation. This data explains the link between reactive oxygen species and sperm toxicity. Given that epigenetic regulation is likely affected in oxidative-stressed spermatozoa, this data show that spermatozoa appear to shut down under these conditions before reaching the egg.

  3. Role of the lipid peroxidation product, 4-hydroxynonenal, in the development of nitrate tolerance.

    PubMed

    D'Souza, Yohan; Kawamoto, Toshihiro; Bennett, Brian M

    2014-04-21

    Tolerance to nitrates such as nitroglycerin (GTN) is associated with oxidative stress, inactivation of aldehyde dehydrogenase 2 (ALDH2), and decreased GTN-induced cGMP accumulation and vasodilation. We hypothesized that GTN-induced inactivation of ALDH2 results in increased 4-hydroxy-2-nonenal (HNE) adduct formation of key proteins involved in GTN bioactivation, and, consequently, an attenuated vasodilator response to GTN (i.e., tolerance). We used an in vivo GTN tolerance model, a cell culture model of nitrate action, and Aldh2(-/-) mice to assess whether GTN exposure resulted in HNE adduct formation, and whether exogenous HNE affected GTN-induced relaxation and cGMP accumulation. Immunoblot analysis indicated a marked increase in HNE adduct formation in GTN-tolerant porcine kidney epithelial cells (PK1) and in aortae from GTN-tolerant rats and untreated Aldh2(-/-) mice. Preincubation of PK1 cells with HNE resulted in a dose-dependent decrease in GTN-induced cGMP accumulation, and pretreatment of isolated rat aorta with HNE resulted in dose-dependent decreases in the vasodilator response to GTN, thus mimicking GTN-tolerance. Pretreatment of aortae from Aldh2(-/-) mice with 10 μM HNE resulted in a desensitized vasodilator response to GTN. In the in vivo rat tolerance model, changes in HNE adduct formation correlated well with the onset of GTN tolerance and tolerance reversal. Furthermore, coadministration of an HNE scavenger during the tolerance induction protocol completely prevented HNE adduct formation and GTN tolerance but did not prevent the inactivation of ALDH2. The data are consistent with a novel mechanism of GTN tolerance suggesting a primary role of HNE adduct formation in the development of GTN tolerance.

  4. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

  5. Routes to 4-Hydroxynonenal: Fundamental Issues in the Mechanisms of Lipid Peroxidation*S⃞

    PubMed Central

    Schneider, Claus; Porter, Ned A.; Brash, Alan R.

    2008-01-01

    Although investigation of the toxicological and physiological actions of α/β-unsaturated 4-hydroxyalkenals has made great progress over the last 2 decades, understanding of the chemical mechanism of formation of 4-hydroxynonenal and related aldehydes has advanced much less. The aim of this review is to discuss mechanistic evidence for these non-enzymatic routes, especially of the underappreciated intermolecular pathways that involve dimerized and oligomerized fatty acid derivatives as key intermediates. These cross-molecular reactions of fatty acid peroxyls have also important implications for understanding of the basic initiation and propagation steps during lipid peroxidation and the nature of the products that arise. PMID:18285327

  6. Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death.

    PubMed

    Awasthi, Yogesh C; Sharma, Rajendra; Sharma, Abha; Yadav, Sushma; Singhal, Sharad S; Chaudhary, Pankaj; Awasthi, Sanjay

    2008-07-15

    Within the last two decades, 4-hydroxynonenal has emerged as an important second messenger involved in the regulation of various cellular processes. Our recent studies suggest that HNE can induce apoptosis in various cells through the death receptor Fas (CD95)-mediated extrinsic pathway as well as through the p53-dependent intrinsic pathway. Interestingly, through its interaction with the nuclear protein Daxx, HNE can self-limit its apoptotic role by translocating Daxx to cytoplasm where it binds to Fas and inhibits Fas-mediated apoptosis. In this paper, after briefly describing recent studies on various biological activities of HNE, based on its interactions with Fas, Daxx, and p53, we speculate on possible mechanisms through which HNE may affect a multitude of cellular processes and draw a parallel between signaling roles of H(2)O(2) and HNE.

  7. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

    SciTech Connect

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

  8. Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

    PubMed Central

    Jang, Eun Ji; Jeong, Hyoung Oh; Park, Daeui; Kim, Dae Hyun; Choi, Yeon Ja; Chung, Ki Wung; Park, Min Hi; Yu, Byung Pal; Chung, Hae Young

    2015-01-01

    4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process. PMID:26466383

  9. Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.

    PubMed

    Fang, Xinqiang; Fu, Yuan; Long, Marcus J C; Haegele, Joseph A; Ge, Eva J; Parvez, Saba; Aye, Yimon

    2013-10-01

    In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

  10. 4-Hydroxynonenal promotes growth and angiogenesis of breast cancer cells through HIF-1α stabilization.

    PubMed

    Li, Yao-Ping; Tian, Fu-Guo; Shi, Peng-Cheng; Guo, Ling-Yun; Wu, Hai-Ming; Chen, Run-Qi; Xue, Jin-Ming

    2014-01-01

    4-Hydroxynonenal (4-HNE) is a stable end product of lipid peroxidation, which has been shown to play an important role in cell signal transduction, while increasing cell growth and differentiation. 4-HNE could inhibit phosphatase and tensin homolog (PTEN) activity in hepatocytes and increased levels have been found in human invasive breast cancer. Here we report that 4-HNE increased the cell growth of breast cancer cells as revealed by colony formation assay. Moreover, vascular endothelial growth factor (VEGF) expression was elevated, while protein levels of hypoxia inducible factor 1 alpha (HIF-1α) were up-regulated. Sirtuin-3 (SIRT3), a major mitochondria NAD+-dependent deacetylase, is reported to destabilize HIF-1α. Here, 4-HNE could inhibit the deacetylase activity of SIRT3 by thiol-specific modification. We further demonstrated that the regulation by 4-HNE of levels of HIF-1α and VEGF depends on SIRT3. Consistent with this, 4-HNE could not increase the cell growth in SIRT3 knockdown breast cancer cells. Additionally, 4-HNE promoted angiogenesis and invasion of breast cancer cells in a SIRT3-dependent manner. In conclusion, we propose that 4-HNE promotes growth, invasion and angiogenesis of breast cancer cells through the SIRT3-HIF-1α-VEGF axis.

  11. Stimulation of HeLa cell growth by physiological concentrations of 4-hydroxynonenal.

    PubMed

    Zarkovic, N; Ilic, Z; Jurin, M; Schaur, R J; Puhl, H; Esterbauer, H

    1993-12-01

    The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimulating effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin-HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 microM), which was primarily cytotoxic and a physiological range (below 10 microM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.

  12. Lipid peroxidation product 4-hydroxynonenal as factor of oxidative homeostasis supporting bone regeneration with bioactive glasses.

    PubMed

    Mrakovcic, Lidija; Wildburger, Renate; Jaganjac, Morana; Cindric, Marina; Cipak, Ana; Borovic-Sunjic, Suzana; Waeg, Georg; Milankovic, Andrea M; Zarkovic, Neven

    2010-01-01

    Bone regeneration is a process of vital importance since fractures of long bones and large joints have a highly deleterious impact on both, individuals and society. Numerous attempts have been undertaken to alleviate this severe medical and social problem by development of novel bioactive materials, among which bioactive glass is the most attractive because of its osteoconductive and osteostimulative properties. Since lipid peroxidation is an important component of systematic stress response in patients with traumatic brain injuries and bone fractures, studies have been undertaken of the molecular mechanisms of the involvement of 4-hydroxynonenal (HNE), an end product of lipid peroxidation, in cellular growth regulation. We found that HNE generated in bone cells grown in vitro on the surfaces of bioactive glasses 45S5 and 13-93. This raises an interesting possibility of combined action of HNE and ionic bioglass dissolution products in enhanced osteogenesis probably through a mitogen-activated protein kinase (MAPK) pathway. While the proposed mechanism still has to be elucidated, the finding of HNE generation on bioglass offers a new interpretation of the osteoinducting mechanisms of bioglass and suggests the possibility of tissue engineering based on manipulations of oxidative homeostasis.

  13. Stobadine attenuates impairment of an intestinal barrier model caused by 4-hydroxynonenal.

    PubMed

    Cindric, Marina; Cipak, Ana; Zapletal, Emilija; Jaganjac, Morana; Milkovic, Lidija; Waeg, Georg; Stolc, Svorad; Zarkovic, Neven; Suzana Borovic, Sunjic

    2013-02-01

    Alterations in the intestinal barrier permeability occur in a broad spectrum of abdominally related pathologies, mostly due to disturbed oxidative homeostasis and increased lipid peroxidation. 4-Hydroxynonenal (HNE), a major lipid peroxidation product, is physiologically present in healthy gastric mucosa, but is increased in early stages of colon cancer and patients with duodenal peptic ulcer. Nevertheless, such supraphysiological levels of HNE have not yet been associated with increased intestinal permeability, even though, as we have described in this paper, they could play important role. In vitro model of intestinal barrier was established by growing Caco-2 cell line on cell culture permeable inserts. The pyridoindole derivative stobadine in hydrophilic and lipophilic form was used for barrier model protection. Both forms of stobadine were able to prevent damaging HNE effects, and reduce generation of reactive oxygen species and permeability of the intestinal barrier. Immunocytochemical analysis has confirmed beneficial effect of stobadine in reducing the formation of HNE-protein conjugates in the cells. Lipophilic form of stobadine proved to be more efficient than hydrophilic, implying importance of lipids in maintaining barrier function. The results obtained indicate that HNE might be important factor affecting intestinal barrier integrity, while stobadine could efficiently protect intestinal cells against harmful HNE effects.

  14. Macrophage uptake of low-density lipoprotein modified by 4-hydroxynonenal. An ultrastructural study

    SciTech Connect

    Hoff, H.F.; Cole, T.B. )

    1991-02-01

    We have documented the ultrastructural characteristics of the uptake and processing by mouse peritoneal macrophages (MPM) of low-density lipoprotein (LDL) modified with 4-hydroxynonenal (HNE), an intermediate of lipid peroxidation. This was performed as part of a larger biochemical study assessing the role of LDL oxidation in lipid loading of macrophages during atherogenesis. Gold-labeled LDL that was modified with HNE leading to particle aggregation represented the morphologic probe used. When incubated with MPM, the probe became associated with short segments of cell membrane, probably derived from blebs or from lysed cells. At 37 degrees C there was a time-dependent increase in uptake by MPM, and at 4 hours the increase paralleled the degradation by MPM of 125I-labeled HNE-LDL-cAu. Clathrin-coated pits on the cell surface were consistently associated with probe. Uptake of probe appeared to occur via phagocytosis, because pseudopods frequently surrounded probe, and cytochalasin D quantitatively prevented probe uptake. A time-dependent increase was found in the number of gold particles per unit area within vacuoles, some of which were secondary lysosomes, based on acid phosphatase-positive staining. Thus, HNE-induced aggregation of LDL during oxidation, binding of aggregates to clathrin-coated pits on MPM, and subsequent phagocytosis may represent one of the ways lipid-laden foam cells are formed in vivo.

  15. Studies on epitopes on low-density lipoprotein modified by 4-hydroxynonenal. Biochemical characterization and determination.

    PubMed Central

    Chen, Q; Esterbauer, H; Jürgens, G

    1992-01-01

    Oxidation of human low-density lipoprotein (LDL) was found to be accompanied by the generation of various reactive aldehydes. One of them, 4-hydroxynonenal (HNE), was shown to modify LDL to a form which represents a good model of oxidized LDL (ox-LDL). In order to investigate the epitopes newly formed on HNE-modified LDL, a polyvalent antiserum to HNE-LDL [anti-(HNE-LDL)] was raised in rabbits and the non-specific components were removed with native LDL coupled to CNBr-Sepharose 4B. Competitive fluorescence immunoassay analysis showed that anti-(HNE-LDL) recognized HNE-LDL, copper-oxidized LDL, HNE-albumin and to a lower extent HNE-modified high-density lipoprotein 3 (HNE-HDL3) and ox-HDL3 but not native LDL. A certain degree of cross-reactivity of the antibody with LDLs modified by either hexanal or 2,4-heptadienal was found. No reaction was obtained with LDL labelled with malondialdehyde. From the abilities of HNE-modified poly(L-amino acids) to compete with HNE-LDL for binding to anti-(HNE-LDL), it is postulated that lysine, tyrosine, arginine and histidine are involved in the formation of HNE-derived epitopes on apolipoprotein B (apo B). Using a double-sandwich fluorescence immunoassay [capture antibody: anti-(apo B); detection antibody: anti-(HNE-LDL)] we found that the HNE-derived epitopes were expressed at a far higher degree in ox-LDL and HNE-LDL than in native LDL. PMID:1280111

  16. 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.

  17. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    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

  18. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide.

    PubMed

    Mark, R J; Lovell, M A; Markesbery, W R; Uchida, K; Mattson, M P

    1997-01-01

    Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid beta-peptide (A beta) can promote free radical production, we tested the hypothesis that HNE mediates A beta 25-35-induced disruption of neuronal ion homeostasis and cell death. A beta induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+, K(+)-ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both A beta and HNE. The antioxidant propyl gallate protected neurons against A beta toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates A beta-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration. PMID:8978733

  19. Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

    PubMed Central

    Dodson, Matthew; Liang, Qiuli; Johnson, Michelle S; Redmann, Matthew; Fineberg, Naomi; Darley-Usmar, Victor M; Zhang, Jianhua

    2013-01-01

    How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE. PMID:24145463

  20. Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance

    PubMed Central

    Dalleau, S; Baradat, M; Guéraud, F; Huc, L

    2013-01-01

    During the last three decades, 4-hydroxy-2-nonenal (HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of pathologies such as metabolic diseases, neurodegenerative diseases and cancers. These multiple pathologies can be explained by the fact that HNE is a potent modulator of numerous cell processes such as oxidative stress signaling, cell proliferation, transformation or cell death. The main objective of this review is to focus on the different aspects of HNE-induced cell death, with a particular emphasis on apoptosis. HNE is a special apoptotic inducer because of its abilities to form protein adducts and to propagate oxidative stress. It can stimulate intrinsic and extrinsic apoptotic pathways and interact with typical actors such as tumor protein 53, JNK, Fas or mitochondrial regulators. At the same time, due to its oxidant status, it can also induce some cellular defense mechanisms against oxidative stress, thus being involved in its own detoxification. These processes in turn limit the apoptotic potential of HNE. These dualities can imbalance cell fate, either toward cell death or toward survival, depending on the cell type, the metabolic state and the ability to detoxify. PMID:24096871

  1. The balance between 4-hydroxynonenal and intrinsic glutathione/glutathione S-transferase A4 system may be critical for the epidermal growth factor receptor phosphorylation of human esophageal squamous cell carcinomas.

    PubMed

    Uno, Kaname; Kato, Katsuaki; Kusaka, Gen; Asano, Naoki; Iijima, Katsunori; Shimosegawa, Tooru

    2011-10-01

    Oxidative stress might participate in the carcinogenesis of human esophageal squamous cell carcinomas (hESCC). 4-Hydroxynonenal (HNE) is a major product of membrane lipid peroxidation with short life. It might act as an important mediator through the generation of adducts and activate epidermal growth factor receptor (EGFR) signaling. It is mainly trapped with glutathione (GSH) and catalyzed by glutathione S-transferases (GSTs). This study aimed to elucidate the possible participation of HNE, GSH/GST system, and EGFR signaling in hESCC development. Immunohistochemistry of HNE adducts, EGFR, and phosphorylated EGFR (pEGFR) was performed with hESCC specimens. The effect of HNE on the phosphorylation of EGFR and its downstream PhospholipaseCγ1 (PLCγ1) was investigated with KYSE30 cell-line. Pretreatment with GSH inducer N-acetylcysteine (NAC) or GSH inhibitor Buthionine sulfoximine (BSO) and mandatory transfection of hGSTA4 gene in KYSE30 were conducted to investigate the relationship between HNE and GSH/GST system. Immunoreactants of HNE adducts, EGFR, and pEGFR were increased in hESCC compared to non-cancerous epithelium with positive correlations. The treatment of HNE ligand-independently induced the phosphorylation of EGFR and PLCγ1 accompanying the diminishment of intracellular GSH level. NAC increased GSH contents but BSO decreased in dose-dependent manners. Reflecting changes in GSH, HNE-induced EGFR phosphorylation was suppressed by NAC, whereas it was promoted by BSO. Mandatory expression of hGSTA4 suppressed HNE-induced events. We first demonstrated that the ligand-independent activation of EGFR by the balance between the stimulation of HNE and the prevention of intrinsic GSH/GST system might participate in the development of hESCC.

  2. Role of the Lipoperoxidation Product 4-Hydroxynonenal in the Pathogenesis of Severe Malaria Anemia and Malaria Immunodepression

    PubMed Central

    Schwarzer, Evelin; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    Oxidative stress plays an important role in the pathogenesis of falciparum malaria, a disease still claiming close to 1 million deaths and 200 million new cases per year. Most frequent complications are severe anemia, cerebral malaria, and immunodepression, the latter being constantly present in all forms of malaria. Complications are associated with oxidative stress and lipoperoxidation. Its final product 4-hydroxynonenal (4-HNE), a stable yet very reactive and diffusible molecule, forms covalent conjugates with proteins, DNA, and phospholipids and modulates important cell functions at very low concentrations. Since oxidative stress plays important roles in the pathogenesis of severe malaria, it appears important to explore the role of 4-HNE in two important malaria complications such as malaria anemia and malaria immunodepression where oxidative stress is considered to be involved. In this review we will summarize data about 4-HNE chemistry, its biologically relevant chemical properties, and its role as regulator of physiologic processes and as pathogenic factor. We will review studies documenting the role of 4-HNE in severe malaria with emphasis on malaria anemia and immunodepression. Data from other diseases qualify 4-HNE both as oxidative stress marker and as pathomechanistically important molecule. Further studies are needed to establish 4-HNE as accepted pathogenic factor in severe malaria. PMID:25969702

  3. Formation of Malondialdehyde, 4-Hydroxynonenal, and 4-Hydroxyhexenal during in Vitro Digestion of Cooked Beef, Pork, Chicken, and Salmon.

    PubMed

    Steppeler, Christina; Haugen, John-Erik; Rødbotten, Rune; Kirkhus, Bente

    2016-01-20

    Red meat high in heme iron may promote the formation of potentially genotoxic aldehydes during lipid peroxidation in the gastrointestinal tract. In this study, the formation of malondialdehyde (MDA) equivalents measured by the thiobarbituric acid reactive substances (TBARS) method was determined during in vitro digestion of cooked red meat (beef and pork), as well as white meat (chicken) and fish (salmon), whereas analysis of 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE) was performed during in vitro digestion of cooked beef and salmon. Comparing products with similar fat contents indicated that the amount of unsaturated fat and not total iron content was the dominating factor influencing the formation of aldehydes. It was also shown that increasing fat content in beef products caused increasing concentrations of MDA equivalents. The highest levels, however, were found in minced beef with added fish oil high in unsaturated fat. This study indicates that when ingested alone, red meat products low in unsaturated fat and low in total fat content contribute to relatively low levels of potentially genotoxic aldehydes in the gastrointestinal tract.

  4. Increased levels of 4-hydroxynonenal and acrolein in the brain in preclinical Alzheimer’s disease (PCAD)

    PubMed Central

    Bradley, M. A.; Markesbery, W. R.

    2010-01-01

    Previous studies demonstrate increased levels of 4-hydroxynonenal (HNE) and acrolein in vulnerable brain regions of subjects with mild cognitive impairment (MCI) and late-stage Alzheimer’s disease (AD). Recently preclinical AD (PCAD) subjects, who demonstrate normal antemortem neuropsychological test scores but abundant AD pathology at autopsy, have become the focus of increased study. Levels of extractable HNE and acrolein were quantified by gas chromatography mass spectrometry with negative chemical ionization and protein-bound HNE and acrolein was quantified by dot-blot immunohistochemistry in the hippocampus/parahippocampal gyrus (HPG), superior and middle temporal gyri (SMTG), and cerebellum (CER) of 10 PCAD and 10 age-matched normal control (NC) subjects. Results of the analyses show a significant (p < 0.05) increase in levels of extractable acrolein in HPG of PCAD subjects compared to age-matched NC subjects and a significant decrease of extractable acrolein in PCAD CER. A significant increase in protein-bound HNE in HPG and a significant decrease in CER of PCAD subjects compared to NC subjects was observed. No significant alterations were observed in either extractable or protein-bound HNE or acrolein in the SMTG of PCAD subjects. Additionally, no significant differences in levels of protein carbonyls were observed in the HPG, SMTG, or CER of PCAD subjects compared to NC subjects. PMID:20171275

  5. Gsta4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity*

    PubMed Central

    McElhanon, Kevin E.; Bose, Chhanda; Sharma, Rajendra; Wu, Liping; Awasthi, Yogesh C.; Singh, Sharda P.

    2013-01-01

    The alpha class glutathione s-transferase (GST) isozyme GSTA4–4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4–4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4–4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4–4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE. PMID:24353929

  6. Serum Concentrations of F2-Isoprostanes and 4-Hydroxynonenal in Hemodialysis Patients in Relation to Inflammation and Renal Anemia

    PubMed Central

    Wiswedel, Ingrid; Peter, Daniela; Gardemann, Andreas; Carluccio, Francesco; Hampl, Hannelore; Siems, Werner

    2008-01-01

    Background Patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD) are apparently exposed to enhanced oxidative stress and to inflammation. It was the aim of this study to characterize the state of systemic oxidative stress of ESRD patients before and following HD using highly specific biomarkers, F2-isoprostanes and 4-hydroxynonenal (HNE). Furthermore the question should be answered, if there are associations between inflammation and systemic oxidative stress and/or between systemic oxidative stress and renal anemia, which is more or less typical for HD patients. Patients and methods Concentrations of F2-isoprostanes, HNE, C-reactive protein (CRP) as marker of inflammation, and hemoglobin were measured in serum samples of patients with ESRD before and after HD and of healthy control persons for comparison. Total (esterified plus free) F2-isoprostanes were quantified by highly sensitive gas chromatography/mass spectrometry technique, HNE by thin layer chromatography and HPLC/UV detection, CRP by immunoturbidimetry and hemoglobin by clinico-chemical routine assay. Results 1. HD patients showed significantly higher serum concentrations of F2-isoprostanes and HNE than healthy human control subjects. 2. Total (esterified plus free) F2-isoprostane levels before HD were not significantly different from those after HD, whereas HNE levels were significantly decreased in patients after HD. 3. F2-isoprostane concentrations in HD patients correlated with the levels of CRP, whereas HNE concentrations inversely correlated with the content of hemoglobin. Conclusion Both, F2-isoprostanes and HNE serum concentrations are useful oxidative stress parameters in ESRD patients undergoing HD. Whereas HNE strongly correlates with the severity of renal anemia, leading to left heart insufficiency, F2-isoprostanes (sum of free plus esterified) highly correlate with the degree of inflammation. PMID:19578522

  7. ACCUMULATION OF M1DG DNA ADDUCTS AFTER CHRONIC EXPOSURE TO PCBS, BUT NOT FROM ACUTE EXPOSURE TO DIOXIN-LIKE COMPOUNDS

    EPA Science Inventory

    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 exposur...

  8. Preferential binding of 4-hydroxynonenal to lysine residues in specific parasite proteins in plakortin-treated Plasmodium falciparum-parasitized red blood cells

    PubMed Central

    Schwarzer, Evelin; Gallo, Valentina; Valente, Elena; Ulliers, Daniela; Taglialatela-Scafati, Orazio; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    The data show the frequencies by which the amino acid residues lysine, histidine and cysteine of six proteins of the malaria parasite Plasmodium falciparum are post-translationally modified by the lipoperoxydation endproduct 4-hydroxynonenal after challenging the parasitized red blood cell with plakortin. Plakortin is an antimalarial endoperoxide whose molecular anti-parasitic effect is described in Skorokhod et al. (2015) [1]. Plakortin did not elicit hemoglobin leakage from host red blood cells and did not oxidize reduced glutathione. PMID:26702418

  9. Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male wild type 129/SvJ mice, and glutathione S-transferase A4-4 null (GSTA4-/-) mice for 40 d. GSTA4-/- mice were also crossed with peroxisome proliferator-activated ...

  10. [The level of 8-iso-prostaglandin F2 alpha, 4-hydroxynonenal and malondialdehyde in alcohol dependent men during combined therapy].

    PubMed

    Kopczyńska, Ewa; Lampka, Magdalena; Torliński, Lech; Ziółkowski, Marcin

    2002-01-01

    The aim of the study was the estimation of intensity of lipid peroxidation in alcohol dependent male patients after three months of therapy with naltrexone or tianeptine and the next three months follow-up. 61 males with clinical diagnosis of alcohol dependence (ICD-10) have been examined. The investigated parameters have been determined in blood serum, the 8-iso-prostaglandin F2 alpha by means of immunoenzymatic assay (ELISA) and malondialdehyde with 4-hydroxynonenal by means of colorimetric method. In alcohol dependent men before pharmacotherapy the mean concentration of 8-iso-PGF2 alpha and [MDA + 4-HNE] was higher than the reference interval. Both, after three months of applied drugs and the next three months follow-up, the concentration of studied parameters decreased considerably. The above results show intensification of lipid peroxidation in alcohol abusers and advantageous influence of abstinence from alcohol and treatment of naltrexone or tianeptine on free-radical changes of lipids as well.

  11. Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death.

    PubMed

    Martin Muñoz, Patricia; Ortega Ferrusola, Cristina; Vizuete, Guillermo; Plaza Dávila, Maria; Rodriguez Martinez, Heriberto; Peña, Fernando J

    2015-12-01

    Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted.

  12. The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard

    SciTech Connect

    Zheng, Ruijin; Po, Iris; Mishin, Vladimir; Black, Adrienne T.; Heck, Diane E.; Laskin, Debra L.; Sinko, Patrick J.; Gerecke, Donald R.; Gordon, Marion K.; Laskin, Jeffrey D.

    2013-10-15

    The cornea is highly sensitive to oxidative stress, a process that can lead to lipid peroxidation. Ultraviolet light B (UVB) and nitrogen mustard (mechlorethamine) are corneal toxicants known to induce oxidative stress. Using a rabbit air-lifted corneal organ culture model, the oxidative stress responses to these toxicants in the corneal epithelium was characterized. Treatment of the cornea with UVB (0.5 J/cm{sup 2}) or nitrogen mustard (100 nmol) resulted in the generation of 4-hydroxynonenal (4-HNE), a reactive lipid peroxidation end product. This was associated with increased expression of the antioxidant, heme oxygenase-1 (HO-1). In human corneal epithelial cells in culture, addition of 4-HNE or 9-nitrooleic acid, a reactive nitrolipid formed during nitrosative stress, caused a time-dependent induction of HO-1 mRNA and protein; maximal responses were evident after 10 h with 30 μM 4-HNE or 6 h with 10 μM 9-nitrooleic acid. 4-HNE and 9-nitrooleic acid were also found to activate Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE- and 9-nitrooleic acid-induced HO-1 expression. Inhibition of Erk1/2, and to a lesser extent, JNK and PI3K/Akt, suppressed only 4-HNE-induced HO-1, while inhibition of JNK and PI3K/Akt, but not Erk1/2, partly reduced 9-nitrooleic acid-induced HO-1. These data indicate that the actions of 4-HNE and 9-nitrooleic acid on corneal epithelial cells are distinct. The sensitivity of corneal epithelial cells to oxidative stress may be an important mechanism mediating tissue injury induced by UVB or nitrogen mustard. - Highlights: • UVB or nitrogen mustard causes rabbit corneal epithelial injury. • 4-Hydroxynonenal (4-HNE) was formed and heme oxygenase-1 (HO-1) was increased. • 4-HNE induced HO-1 mRNA and protein expression in human corneal epithelial cells. • The induction of HO-1 by 4-HNE was through MAP kinase activation.

  13. Alterations in mitochondrial respiratory functions, redox metabolism and apoptosis by oxidant 4-hydroxynonenal and antioxidants curcumin and melatonin in PC12 cells

    SciTech Connect

    Raza, Haider John, Annie; Brown, Eric M.; Benedict, Sheela; Kambal, Amr

    2008-01-15

    Cellular oxidative stress and alterations in redox metabolisms have been implicated in the etiology and pathology of many diseases including cancer. Antioxidant treatments have been proven beneficial in controlling these diseases. We have recently shown that 4-hydroxynonenal (4-HNE), a by-product of lipid peroxidation, induces oxidative stress in PC12 cells by compromising the mitochondrial redox metabolism. In this study, we have further investigated the deleterious effects of 4-HNE on mitochondrial respiratory functions and apoptosis using the same cell line. In addition, we have also compared the effects of two antioxidants, curcumin and melatonin, used as chemopreventive agents, on mitochondrial redox metabolism and respiratory functions in these cells. 4-HNE treatment has been shown to cause a reduction in glutathione (GSH) pool, an increase in reactive oxygen species (ROS), protein carbonylation and apoptosis. A marked inhibition in the activities of the mitochondrial respiratory enzymes, cytochrome c oxidase and aconitase was observed after 4-HNE treatment. Increased nuclear translocation of NF-kB/p65 protein was also observed after 4-HNE treatment. Curcumin and melatonin treatments, on the other hand, maintained the mitochondrial redox and respiratory functions without a marked effect on ROS production and cell viability. These results suggest that 4-HNE-induced cytotoxicity may be associated, at least in part, with the altered mitochondrial redox and respiratory functions. The alterations in mitochondrial energy metabolism and redox functions may therefore be critical in determining the difference between cell death and survival.

  14. Improved Anti-Tumoral Therapeutic Efficacy of 4-Hydroxynonenal Incorporated in Novel Lipid Nanocapsules in 2D and 3D Models.

    PubMed

    Pizzimenti, Stefania; Daga, Martina; Ciamporcero, Eric; Toaldo, Cristina; Pettazzoni, Piergiorgio; Osella-Abate, Simona; Novelli, Mauro; Minelli, Rosalba; Bisazza, Agnese; Gamba, Paola; Testa, Gabriella; Ullio, Chiara; Ferruti, Paolo; Ranucci, Elisabetta; Bernengo, Maria Grazia; Ferretti, Carlo; Dianzani, Chiara; Biasi, Fiorella; Barrera, Giuseppina; Cavalli, Roberta

    2015-12-01

    4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A β-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments. PMID:26510311

  15. Rapid liquid chromatography-tandem mass spectrometry analysis of 4-hydroxynonenal for the assessment of oxidative degradation and safety of vegetable oils.

    PubMed

    Gabbanini, Simone; Matera, Riccardo; Valvassori, Alice; Valgimigli, Luca

    2015-04-15

    A novel method for the UHPLC-MS/MS analysis of (E)-4-hydroxynonenal (4-HNE) is described. The method is based on derivatization of 4-HNE with pentafluorophenylhydrazine (1) or 4-trifluoromethylphenylhydrazine (2) in acetonitrile in the presence of trifluoroacetic acid as catalyst at room temperature and allows complete analysis of one sample of vegetable oil in only 21 min, including sample preparation and chromatography. The method involving hydrazine 1, implemented in an ion trap instrument with analysis of the transition m/z 337→154 showed LOD=10.9 nM, average accuracy of 101% and precision ranging 2.5-4.0% RSD intra-day (2.7-4.1% RSD inter-day), with 4-HNE standard solutions. Average recovery from lipid matrices was 96.3% from vaseline oil, 91.3% from sweet almond oil and 105.3% from olive oil. The method was tested on the assessment of safety and oxidative degradation of seven samples of dietary oil (soybean, mixed seeds, corn, peanut, sunflower, olive) and six cosmetic-grade oils (avocado, blackcurrant, apricot kernel, echium, sesame, wheat germ) and effectively detected increased 4-HNE levels in response to chemical (Fenton reaction), photochemical, or thermal stress and aging, aimed at mimicking typical oxidation associated with storage or industrial processing. The method is a convenient, cost-effective and reliable tool to assess quality and safety of vegetable oils.

  16. 4-Hydroxynonenal, an aldehydic product of lipid peroxidation, impairs signal transduction associated with muscarinic acetylcholine and metabotropic glutamate receptors: possible action on G alpha(q/11).

    PubMed

    Blanc, E M; Kelly, J F; Mark, R J; Waeg, G; Mattson, M P

    1997-08-01

    Considerable data indicate that oxidative stress and membrane lipid peroxidation contribute to neuronal degeneration in an array of age-related neurodegenerative disorders. In contrast, the impact of subtoxic levels of membrane lipid peroxidation on neuronal function is largely unknown. We now report that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, disrupts coupling of muscarinic cholinergic receptors and metabotropic glutamate receptors to phospholipase C-linked GTP-binding proteins in cultured rat cerebrocortical neurons. At subtoxic concentrations, HNE markedly inhibited GTPase activity, inositol phosphate release, and elevation of intracellular calcium levels induced by carbachol (muscarinic agonist) and (RS)-3,5-dihydroxyphenyl glycine (metabotropic glutamate receptor agonist). Maximal impairment of agonist-induced responses occurred within 30 min of exposure to HNE. Other aldehydes, including malondialdehyde, had little effect on agonist-induced responses. Antioxidants that suppress lipid peroxidation did not prevent impairment of agonist-induced responses by HNE, whereas glutathione, which is known to bind and detoxify HNE, did prevent impairment of agonist-induced responses. HNE itself did not induce oxidative stress. Immunoprecipitation-western blot analysis using an antibody to HNE-protein conjugates showed that HNE can bind to G alpha(q/11). HNE also significantly suppressed inositol phosphate release induced by aluminum fluoride. Collectively, our data suggest that HNE plays a role in altering receptor-G protein coupling in neurons under conditions of oxidative stress that may occur both normally, and before cell degeneration and death in pathological settings. PMID:9231714

  17. γ-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells

    PubMed Central

    Zhang, Hongqiao; Liu, Honglei; Dickinson, Dale A.; Liu, Rui-Ming; Postlethwait, Edward M.; Laperche, Yannick; Forman, Henry Jay

    2009-01-01

    γ-Glutamyl transpeptidase (GGT) plays key roles in glutathione homeostasis and metabolism of glutathione S-conjugates. Rat GGT is transcribed via five tandemly arranged promoters into seven transcripts. The transcription of mRNAV is controlled by promoter 5. Previously we found that GGT mRNAV-2 was responsible for the induction of GGT in rat alveolar epithelial cells by 4-hydroxynonenal (HNE). In the current study, the underlying mechanism was investigated. Reporter deletion and mutation analysis demonstrated that an electrophile-response element (EpRE) in the proximal region of GGT promoter 5 (GP5) was responsible for the basal- and HNE-induced promoter activity. Gel-shift assays showed an increased binding activity of GP5 EpRE after HNE exposure. The nuclear content of NF-E2-related factor 2 (Nrf2) was significantly increased by HNE. The recruitment of Nrf2 to GP5 EpRE after HNE treatment was demonstrated by supershift and chromatin immunoprecipitation assays. The tissue expression pattern of GGT mRNA V was previously unknown. Using polymerase chain reaction, we found that GGT mRNAV-2 was expressed in many tissues in rat. Taken together, GGT mRNAV-2 is widely expressed in rat tissues and its basal and HNE-induced expression is mediated through EpRE/Nrf2 signaling. PMID:16631518

  18. Influence of 4-hydroxynonenal and spleen cells on primary hepatocyte culture and a novel liver-derived cell line resembling hepatocyte stem cells.

    PubMed

    Cipak, Ana; Borovic, Suzana; Jaganjac, Morana; Bresgen, Nikolaus; Kirac, Iva; Grbesa, Ivana; Mrakovcic, Lidija; Cindric, Marina; Scukanec-Spoljar, Mira; Gall-Troselj, Koraljka; Coric, Marijana; Eckl, Peter; Zarkovic, Neven

    2010-01-01

    Liver is a unique mammalian organ with a great capacity of regeneration related to its function. After surgical resection or injury, hepatic cells, especially hepatocytes, can proliferate rapidly to repair the damage and to regenerate the structure without affecting the function of the liver. Loss of catalase activity during regeneration indicates that oxidative stress is present in the liver not only in pathological conditions but also as a 'physiological' factor during regeneration. As we have shown in our previous work, liver stem cell-like cells treated with 4-hydroxynonenal (HNE), a cytotoxic and growth regulating lipid peroxidation product, recover in the presence of spleen cells. In the current study we characterized this novel cell line as liver-derived progenitor/oval-like cells, (LDP/OCs), i.e. functional liver stem-like cells. We showed that LDP/OC were OV6 positive, with abundant glycogen content in the cytoplasm and expressed alpha-fetoprotein, albumin, biliverdin reductase and gamma-glutamyl transferase. Also, we compared their growth in vitro with the growth of cultured primary hepatocytes stressed with HNE and co-cultured with autologous spleen cells. The influence of spleen cells on HNE-treated primary hepatocytes and on LDP/OCs showed that spleen cells support in a similar manner the recovery of both types of liver cells indicating their important role in regeneration. Hence, LDP/OC cells may provide a valuable tool to study cell interactions and the role on HNE in liver regeneration.

  19. Rapid liquid chromatography-tandem mass spectrometry analysis of 4-hydroxynonenal for the assessment of oxidative degradation and safety of vegetable oils.

    PubMed

    Gabbanini, Simone; Matera, Riccardo; Valvassori, Alice; Valgimigli, Luca

    2015-04-15

    A novel method for the UHPLC-MS/MS analysis of (E)-4-hydroxynonenal (4-HNE) is described. The method is based on derivatization of 4-HNE with pentafluorophenylhydrazine (1) or 4-trifluoromethylphenylhydrazine (2) in acetonitrile in the presence of trifluoroacetic acid as catalyst at room temperature and allows complete analysis of one sample of vegetable oil in only 21 min, including sample preparation and chromatography. The method involving hydrazine 1, implemented in an ion trap instrument with analysis of the transition m/z 337→154 showed LOD=10.9 nM, average accuracy of 101% and precision ranging 2.5-4.0% RSD intra-day (2.7-4.1% RSD inter-day), with 4-HNE standard solutions. Average recovery from lipid matrices was 96.3% from vaseline oil, 91.3% from sweet almond oil and 105.3% from olive oil. The method was tested on the assessment of safety and oxidative degradation of seven samples of dietary oil (soybean, mixed seeds, corn, peanut, sunflower, olive) and six cosmetic-grade oils (avocado, blackcurrant, apricot kernel, echium, sesame, wheat germ) and effectively detected increased 4-HNE levels in response to chemical (Fenton reaction), photochemical, or thermal stress and aging, aimed at mimicking typical oxidation associated with storage or industrial processing. The method is a convenient, cost-effective and reliable tool to assess quality and safety of vegetable oils. PMID:25818139

  20. Alterations in mitochondrial respiratory functions, redox metabolism and apoptosis by oxidant 4-hydroxynonenal and antioxidants curcumin and melatonin in PC12 cells.

    PubMed

    Raza, Haider; John, Annie; Brown, Eric M; Benedict, Sheela; Kambal, Amr

    2008-01-15

    Cellular oxidative stress and alterations in redox metabolisms have been implicated in the etiology and pathology of many diseases including cancer. Antioxidant treatments have been proven beneficial in controlling these diseases. We have recently shown that 4-hydroxynonenal (4-HNE), a by-product of lipid peroxidation, induces oxidative stress in PC12 cells by compromising the mitochondrial redox metabolism. In this study, we have further investigated the deleterious effects of 4-HNE on mitochondrial respiratory functions and apoptosis using the same cell line. In addition, we have also compared the effects of two antioxidants, curcumin and melatonin, used as chemopreventive agents, on mitochondrial redox metabolism and respiratory functions in these cells. 4-HNE treatment has been shown to cause a reduction in glutathione (GSH) pool, an increase in reactive oxygen species (ROS), protein carbonylation and apoptosis. A marked inhibition in the activities of the mitochondrial respiratory enzymes, cytochrome c oxidase and aconitase was observed after 4-HNE treatment. Increased nuclear translocation of NF-kB/p65 protein was also observed after 4-HNE treatment. Curcumin and melatonin treatments, on the other hand, maintained the mitochondrial redox and respiratory functions without a marked effect on ROS production and cell viability. These results suggest that 4-HNE-induced cytotoxicity may be associated, at least in part, with the altered mitochondrial redox and respiratory functions. The alterations in mitochondrial energy metabolism and redox functions may therefore be critical in determining the difference between cell death and survival. PMID:17935746

  1. Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal.

    PubMed

    Doi, Adriana M; Pham, Robert T; Hughes, Erin M; Barber, David S; Gallagher, Evan P

    2004-06-01

    We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass (Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione S-transferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis-Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5' and 3' rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice (Pleuronectes platessa), European flounder (Platichthys flesus) and fathead minnow (Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6 x His-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms.

  2. Elevation of 4-hydroxynonenal and malondialdehyde modified protein levels in cerebral cortex with cognitive dysfunction in rats exposed to 1-bromopropane.

    PubMed

    Zhong, Zhixia; Zeng, Tao; Xie, Keqin; Zhang, Cuili; Chen, Jingjing; Bi, Ye; Zhao, Xiulan

    2013-04-01

    1-Bromopropane (1-BP), an alternative to ozone-depleting solvents (ODS), exhibits central nervous system (CNS) toxicity in animals and humans. This study was designed to relate CNS damage by Morris water maze (MWM) test and oxidative stress to 1-BP exposure in the rat. Male Wistar rats were randomly divided into 4 groups (n=10), and treated with 0, 200, 400 and 800 mg/kgbw 1-BP for consecutive 12 days, respectively. From day 8 to day 12 of the experiment, MWM test was employed to assess the cognitive function of rats. The cerebral cortex of rats was obtained immediately following the 24h after MWM test conclusion. Glutathione (GSH), oxidized glutathione (GSSG) and total thiol (total-SH) content, GSH reductase (GR) and GSH peroxidase (GSH-Px) activities, malondialdehyde (MDA) level, as well as 4-hydroxynonenal (4-HNE) and MDA modified proteins in homogenates of cerebral cortex were measured. The obtained results showed that 1-BP led to cognitive dysfunction of rats, which was evidenced by delayed escape latency time and swimming distances in MWM performance. GSH and total-SH content, GSH/GSSG ratio, GR activity significantly decreased in cerebral cortex of rats, coupling with the increase of MDA level. 4-HNE and MDA modified protein levels obviously elevated after 1-BP exposure. GSH-Px activities in cerebral cortex of rats also increased. These data suggested that 1-BP resulted in enhanced lipid peroxidation of brain, which might play an important role in CNS damage induced by 1-BP.

  3. 4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

    SciTech Connect

    Lee, Seung J.; Kim, Chae E.; Yun, Mi R.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Shin, Hwa K.; Bae, Sun S.; Kim, Chi D.

    2010-01-15

    Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B{sub 4} (LTB{sub 4}) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT{sub 1} (cysLT{sub 1}) receptor antagonist, REV-5901 as well as a BLT{sub 1} receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB{sub 4} and cysLT (LTC{sub 4} and LTD{sub 4}) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB{sub 4} and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.

  4. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    PubMed

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    though expression of XPC and CSA were induced by exposure to UVB or pesticides, XPA was the limiting factor in the NER pathway. Our results suggest that pesticides may increase the accumulation of UVB-induced DNA photo-adducts and one likely mechanism is the alteration of critical NER gene expression. The present study provides important implications for evaluating the combined risks of pesticide usage and potentially increasing UVB radiation in aquatic ecosystems.

  5. Association of end-product adducts with increased IgE binding of roasted peanuts.

    PubMed

    Chung, S Y; Champagne, E T

    2001-08-01

    Recently, we have shown that roasted peanuts have a higher level of IgE binding (i.e., potentially more allergenic) than raw peanuts. We hypothesized that this increase in IgE binding of roasted peanuts is due to an increased levels of protein-bound end products or adducts such as advanced glycation end products (AGE), N-(carboxymethyl)lysine (CML), malondialdehyde (MDA), and 4-hydroxynonenal (HNE). To support our hypothesis, we produced polyclonal antibodies (IgG) to each of these adducts, determined their levels in raw and roasted peanuts, and examined their ability to bind to IgE from a pooled serum of patients with clinically important peanut allergy. Results showed that AGE, CML, MDA, and HNE adducts were all present in raw and roasted peanuts. Roasted peanuts exhibited a higher level of AGE and MDA adducts than raw peanuts. IgE was partially inhibited in a competitive ELISA by antibodies to AGE but not by antibodies to CML, MDA, or HNE. This indicates that IgE has an affinity for peanut AGE adducts. Roasted peanuts exhibited a higher level of IgE binding, which was correlated with a higher level of AGE adducts. We concluded that there is an association between AGE adducts and increased IgE binding (i.e., allergenicity) of roasted peanuts.

  6. Effects of toxicologically relevant xenobiotics and the lipid-derived electrophile 4-hydroxynonenal on macrophage cholesterol efflux: silencing carboxylesterase 1 has paradoxical effects on cholesterol uptake and efflux.

    PubMed

    Ross, Matthew K; Borazjani, Abdolsamad; Mangum, Lee C; Wang, Ran; Crow, J Allen

    2014-10-20

    Cholesterol cycles between free cholesterol (unesterified) found predominantly in membranes and cholesteryl esters (CEs) stored in cytoplasmic lipid droplets. Only free cholesterol is effluxed from macrophages via ATP-binding cassette (ABC) transporters to extracellular acceptors. Carboxylesterase 1 (CES1), proposed to hydrolyze CEs, is inactivated by oxon metabolites of organophosphorus pesticides and by the lipid electrophile 4-hydroxynonenal (HNE). We assessed the ability of these compounds to reduce cholesterol efflux from foam cells. Human THP-1 macrophages were loaded with [(3)H]-cholesterol/acetylated LDL and then allowed to equilibrate to enable [(3)H]-cholesterol to distribute into its various cellular pools. The cholesterol-engorged cells were then treated with toxicants in the absence of cholesterol acceptors for 24 h, followed by a 24 h efflux period in the presence of toxicant. A concentration-dependent reduction in [(3)H]-cholesterol efflux via ABCA1 (up to 50%) was found for paraoxon (0.1-10 μM), whereas treatment with HNE had no effect. A modest reduction in [(3)H]-cholesterol efflux via ABCG1 (25%) was found after treatment with either paraoxon or chlorpyrifos oxon (10 μM each) but not HNE. No difference in efflux rates was found after treatments with either paraoxon or HNE when the universal cholesterol acceptor 10% (v/v) fetal bovine serum was used. When the re-esterification arm of the CE cycle was disabled in foam cells, paraoxon treatment increased CE levels, suggesting the neutral CE hydrolysis arm of the cycle had been inhibited by the toxicant. However, paraoxon also partially inhibited lysosomal acid lipase, which generates cholesterol for efflux, and reduced the expression of ABCA1 protein. Paradoxically, silencing CES1 expression in macrophages did not affect the percent of [(3)H]-cholesterol efflux. However, CES1 mRNA knockdown markedly reduced cholesterol uptake by macrophages, with SR-A and CD36 mRNA reduced 3- and 4-fold

  7. Detection of carcinogenic etheno-DNA adducts in children and adolescents with non-alcoholic steatohepatitis (NASH)

    PubMed Central

    Teufel, Ulrike; Peccerella, Teresa; Engelmann, Guido; Bruckner, Thomas; Flechtenmacher, Christa; Millonig, Gunda; Stickel, Felix; Hoffmann, Georg F.; Schirmacher, Peter; Mueller, Sebastian; Bartsch, Helmut

    2015-01-01

    Background Carcinogenic exocyclic-DNA adducts like 1,N6-etheno-2'-deoxyadenosine (εdA) are formed through reactive intermediates of 4-hydroxynonenal (4-HNE) or other lipid peroxidation (LPO) products with the DNA bases A, C, methyl-C and G. High levels of hepatic etheno-DNA adducts have been detected in cancer prone liver diseases including alcoholic liver disease (ALD). In ALD εdA levels correlated significantly with cytochrome P-450 2E1 (CYP2E1) expression which is also induced in non-alcoholic steatohepatitis (NASH). We investigated the occurrence of εdA adducts in children with NASH as a DNA damage marker. Methods Liver biopsies from 21 children/adolescents with histologically proven NASH were analysed for hepatic fat content, inflammation, and fibrosis. εdA levels in DNA, CYP2E1-expression and protein bound 4-hydroxynonenal (HNE) were semi-quantitatively evaluated by immunohistochemistry. Results Among 21 NASH children, εdA levels in the liver were high in 3, moderate in 5, weak in 9 and not elevated in 4 patients. There was a positive correlation between CYP2E1 and protein-bound 4-HNE (r=0.60; P=0.008) and a trend for a positive relationship for CYP2E1 vs. staining intensity of εdA (r=0.45; P=0.06). Inflammatory activity and fibrosis correlated significantly (r=0.49, P=0.023). Conclusions Our results demonstrate for the first time the presence of elevated carcinogenic etheno-DNA lesions (εdA) in the majority (17/21) of liver biopsies from young NASH patients. Our data suggest that LPO-derived etheno-adducts are implicated in NASH. Whether these adducts may serve as predictive risk markers in NASH children to develop hepatocellular cancer later in life remains to be investigated. PMID:26734629

  8. Distribution and time-course of 4-hydroxynonenal, heat shock protein 110/105 family members and cyclooxygenase-2 expression in the hippocampus of rat during trimethyltin-induced neurodegeneration.

    PubMed

    Corvino, V; Marchese, E; Zarkovic, N; Zarkovic, K; Cindric, M; Waeg, G; Michetti, F; Geloso, M C

    2011-08-01

    Trimethyltin (TMT), an organotin compound considered a useful tool to obtain an experimental model of neurodegeneration, exhibits neurotoxicant effects selectively localised in the limbic system and especially in the hippocampus, which are different in the rat and in mice. In the rat hippocampus, we investigated the expression of aldehyde 4-hydroxynonenal, a major bioactive marker of membrane lipid peroxidation, heat shock protein (HSP) 110/105 family members, markers of oxidative stress, and the neuroinflammatory marker cyclooxygenase-2 after TMT-intoxication at various time points after treatment. Our data show that TMT-induced neurodegeneration in the rat hippocampus is associated specifically with oxidative stress and lipid peroxidation, but not with HSP expression, indicating species-specific differences in the neurotoxicity of TMT between rats and mice.

  9. Formation and accumulation of pyridyloxobutyl DNA adducts in F344 rats chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.

    PubMed

    Lao, Yanbin; Yu, Nanxiong; Kassie, Fekadu; Villalta, Peter W; Hecht, Stephen S

    2007-02-01

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 2) are both potent pulmonary carcinogens in rats. The metabolism of NNK to NNAL is stereoselective and reversible, with (S)-NNAL being the major enantiomer formed from NNK. In rats, (R)-NNAL undergoes facile glucuronidation and is rapidly excreted in urine, whereas (S)-NNAL is preferentially retained in tissues and converted to NNK. We hypothesized that the lung carcinogenicity of NNK in the rat is due in part to the preferential retention of (S)-NNAL in the lung, the reconversion to NNK, and then the metabolic activation of NNK to pyridyloxobutyl (POB)-DNA adducts. We tested this hypothesis by treating male F344 rats with 10 ppm of NNK, (R)-NNAL, or (S)-NNAL in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in liver and lung DNA were quantified by HPLC-ESI-MS/MS. At each time point, total adduct levels were higher in the lung than in the liver. O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 13) was the major adduct detected. Total adduct levels in the rats treated with (S)-NNAL were 0.6-1.3 times as great as those in the NNK group in the lung and 0.7-1.4 times in the liver, and 6-14 times higher than those in the (R)-NNAL group in the lung and 11-17 times in the liver. These results suggest that (S)-NNAL is stereoselectively retained in tissues. This study demonstrates for the first time the accumulation and persistence of specific POB-DNA adducts in the rat lung and liver during chronic treatment with NNK, (R)-NNAL, and (S)-NNAL and supports the hypothesis that the preferential retention of (S)-NNAL in the lung, followed by reconversion to NNK and then the metabolic activation of NNK is critical for lung carcinogenesis by NNK and NNAL.

  10. The generation of carcinogenic etheno-DNA adducts in the liver of patients with nonalcoholic fatty liver disease

    PubMed Central

    Linhart, Kirsten-Berit; Glassen, Katharina; Peccerella, Teresa; Waldherr, Rüdiger; Linhart, Heinz; Bartsch, Helmut

    2015-01-01

    Background Nonalcoholic fatty liver disease (NAFLD), in particular its more aggressive form nonalcoholic steatohepatitis (NASH) is increasingly observed as a cause of end stage liver disease and hepatocellular carcinoma (HCC). Reactive oxygen species (ROS) are an important factor in the pathogenesis of HCC. ROS can react with polyunsaturated fatty acids derived from membrane phospholipids resulting in the production of reactive aldehydes as lipid oxidation (LPO) byproducts, such as 4-hydroxynonenal (4 HNE). 4 HNE can react with DNA to form mutagenic exocyclic etheno-DNA adducts. ROS is induced by inflammatory processes, but also by induction of cytochrome P450 2E1 (CYP2E1), as seen with chronic alcohol consumption. Methods Immunohistochemical detection of CYP2E1, 4 HNE and hepatic exocyclic etheno-DNA adducts was performed on liver sections from 39 patients with NFLD. Spearman rank correlation was calculated to examine possible correlations. Results Exocyclic etheno-DNA adducts were detected and correlated significantly with 4 HNE, but not with CYP2E1. Conclusions This is the first description of highly carcinogenic exocyclic etheno-DNA adducts in NAFLD patients. We could show that exocyclic etheno-DNA adducts significantly correlated with lipid peroxidation product 4 HNE, but not with CYP2E1, implying that in NAFLD ROS generation with consecutive DNA damage is rather inflammation driven through various cytokines than by induction of CYP2E1. PMID:26005678

  11. 4-Hydroxynonenal induces mitochondrial oxidative stress, apoptosis and expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells

    SciTech Connect

    Raza, Haider . E-mail: h.raza@uaeu.ac.ae; John, Annie

    2006-10-15

    An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.

  12. Involvement of peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in BDNF signaling during aging and in Alzheimer disease: possible role of 4-hydroxynonenal (4-HNE).

    PubMed

    Benedetti, Elisabetta; D'Angelo, Barbara; Cristiano, Loredana; Di Giacomo, Erica; Fanelli, Francesca; Moreno, Sandra; Cecconi, Francesco; Fidoamore, Alessia; Antonosante, Andrea; Falcone, Roberta; Ippoliti, Rodolfo; Giordano, Antonio; Cimini, Annamaria

    2014-01-01

    Aging and many neurological disorders, such as AD, are linked to oxidative stress, which is considered the common effector of the cascade of degenerative events. In this phenomenon, reactive oxygen species play a fundamental role in the oxidative decomposition of polyunsaturated fatty acids, resulting in the formation of a complex mixture of aldehydic end products, such as malondialdehyde, 4-hydroxynonenal, and other alkenals. Interestingly, 4-HNE has been indicated as an intracellular agonist of peroxisome proliferator-activated receptor β/δ. In this study, we examined, at early and advanced AD stages (3, 9, and 18 months), the pattern of 4-HNE and its catabolic enzyme glutathione S-transferase P1 in relation to the expression of PPARβ/δ, BDNF signaling, as mRNA and protein, as well as on their pathological forms (i.e., precursors or truncated forms). The data obtained indicate a novel detrimental age-dependent role of PPAR β/δ in AD by increasing pro-BDNF and decreasing BDNF/TrkB survival pathways, thus pointing toward the possibility that a specific PPARβ/δ antagonist may be used to counteract the disease progression. PMID:24621497

  13. Copper Accumulation and Lipid Oxidation Precede Inflammation and Myelin Lesions in N,N-Diethyldithiocarbamate Peripheral Myelinopathy

    PubMed Central

    Viquez, Olga M.; Valentine, Holly L.; Amarnath, Kalyani; Milatovic, Dejan; Valentine, William M.

    2008-01-01

    Dithiocarbamates have a wide spectrum of applications in industry, agriculture and medicine with new applications being actively investigated. One adverse effect of dithiocarbamates is the neurotoxicity observed in humans and experimental animals. Results from previous studies have suggested that dithiocarbamates elevate copper and promote lipid oxidation within myelin membranes. In the current study, copper levels, lipid oxidation, protein oxidative damage and markers of inflammation were monitored as a function of N,N-diethyldithiocarbmate (DEDC) exposure duration in an established model for DEDC-mediated myelinopathy in the rat. Intraabdominal administration of DEDC was performed using osmotic pumps for periods of 2, 4, and 8 weeks. Metals in brain, liver and tibial nerve were measured using ICP-MS and lipid oxidation assessed through HPLC measurement of malondialdehyde in tibial nerve, and GC/MS measurement of F2 isoprostanes in sciatic nerve. Protein oxidative injury of sciatic nerve proteins was evaluated through quantification of 4-hydroxynonenal protein adducts using immunoassay, and inflammation monitored by quantifying levels of IgGs and activated macrophages using immunoassay and immunhistochemistry methods, respectively. Changes in these parameters were then correlated to the onset of structural lesions, determined by light and electron microscopy, to delineate the temporal relationship of copper accumulation and oxidative stress in peripheral nerve to the onset of myelin lesions. The data provide evidence that DEDC mediates lipid oxidation and elevation of total copper in peripheral nerve well before myelin lesions or activated macrophages are evident. This relationship is consistent with copper-mediated oxidative stress contributing to the myelinopathy. PMID:18284930

  14. Copper accumulation and lipid oxidation precede inflammation and myelin lesions in N,N-diethyldithiocarbamate peripheral myelinopathy

    SciTech Connect

    Viquez, Olga M.; Valentine, Holly L.; Amarnath, Kalyani; Milatovic, Dejan; Valentine, William M.

    2008-05-15

    Dithiocarbamates have a wide spectrum of applications in industry, agriculture and medicine with new applications being actively investigated. One adverse effect of dithiocarbamates is the neurotoxicity observed in humans and experimental animals. Results from previous studies have suggested that dithiocarbamates elevate copper and promote lipid oxidation within myelin membranes. In the current study, copper levels, lipid oxidation, protein oxidative damage and markers of inflammation were monitored as a function of N,N-diethyldithiocarbamate (DEDC) exposure duration in an established model for DEDC-mediated myelinopathy in the rat. Intra-abdominal administration of DEDC was performed using osmotic pumps for periods of 2, 4, and 8 weeks. Metals in brain, liver and tibial nerve were measured using ICP-MS and lipid oxidation assessed through HPLC measurement of malondialdehyde in tibial nerve, and GC/MS measurement of F{sub 2} isoprostanes in sciatic nerve. Protein oxidative injury of sciatic nerve proteins was evaluated through quantification of 4-hydroxynonenal protein adducts using immunoassay, and inflammation monitored by quantifying levels of IgGs and activated macrophages using immunoassay and immunohistochemistry methods, respectively. Changes in these parameters were then correlated to the onset of structural lesions, determined by light and electron microscopy, to delineate the temporal relationship of copper accumulation and oxidative stress in peripheral nerve to the onset of myelin lesions. The data provide evidence that DEDC mediates lipid oxidation and elevation of total copper in peripheral nerve well before myelin lesions or activated macrophages are evident. This relationship is consistent with copper-mediated oxidative stress contributing to the myelinopathy.

  15. The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4

    SciTech Connect

    Vatsyayan, Rit; Lelsani, Poorna Chandra Rao; Chaudhary, Pankaj; Kumar, Sushil; Awasthi, Sanjay; Awasthi, Yogesh C.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Low concentration of HNE (0.1-1.0 {mu}M) induced secretion of VEGF in RPE cells. Black-Right-Pointing-Pointer VEGF secreted medium of RPE cells promoted proliferation of endothelial cells. Black-Right-Pointing-Pointer VEGFR2 expression was attenuated with increasing concentrations of HNE. Black-Right-Pointing-Pointer These effects of HNE could be blocked by the over expression of GSTA4-4 in cells. -- Abstract: It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 {mu}M HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 {mu}M HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 {mu}M HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce

  16. 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

  17. Polychlorinated Biphenyls Induce Oxidative DNA Adducts in Female Sprague-Dawley Rats.

    PubMed

    Mutlu, Esra; Gao, Lina; Collins, Leonard B; Walker, Nigel J; Hartwell, Hadley J; Olson, James R; Sun, Wei; Gold, Avram; Ball, Louise M; Swenberg, James A

    2016-08-15

    Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N(6)-ethenodeoxyadenosine (1,N(6)-εdA), N(2),3-ethenoguanine (N(2),3-εG), 1,N(2)-ethenodeoxyguanosine (1,N(2)-εdG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N(6)-εdA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 μg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 μg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, and M1dG. Exposure to PCB 153 (1000 μg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N(6)-εdA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, 1,N(2)-εdG, and N(2),3-εG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and

  18. DNA adducts in bronchial biopsies.

    PubMed

    Dunn, B P; Vedal, S; San, R H; Kwan, W F; Nelems, B; Enarson, D A; Stich, H F

    1991-06-19

    To investigate the feasibility of measuring DNA-carcinogen adducts in the lungs of non-surgical patients, endobronchial biopsies were obtained from 78 patients undergoing routine diagnostic bronchoscopy. Lung cancer was present in 37 (47%) of the patients. DNA was isolated from the tissues and analyzed by HPLC- or nuclease-PI-enriched 32P-postlabelling, using procedures selective for aromatic adducts. Chromatograms from all 28 current smokers showed a distinctive diagonal adduct zone which was present in only 24 of 40 ex-smokers and 4 of 10 lifetime non-smokers. Adduct levels and chromatographic patterns were similar in bronchial tissue from different lobes of the lung, in bronchial and alveolar tissue, and in tumor and non-tumor bronchial tissue taken from the same subject. Bronchial DNA adduct levels were strongly associated with cigarette smoking status and dropped rapidly after smoking ceased. Higher levels of DNA adducts seen in the lung-cancer patients were mainly due to cigarette smoking. Frequent alcohol intake was the only dietary factor associated with higher levels of bronchial DNA adducts. We conclude that the level of bronchial DNA adducts is strongly associated with cigarette-smoking history and with alcohol intake, but is not associated with lung cancer independently from its relation to smoking. The results indicate the feasibility of using 32P-postlabelling to detect and quantitate genetic damage in bronchial biopsy specimens.

  19. On the role of 4-hydroxynonenal in health and disease.

    PubMed

    Csala, Miklós; Kardon, Tamás; Legeza, Balázs; Lizák, Beáta; Mandl, József; Margittai, Éva; Puskás, Ferenc; Száraz, Péter; Szelényi, Péter; Bánhegyi, Gábor

    2015-05-01

    Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,β-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

  20. DNA adducts-chemical addons

    PubMed Central

    Rajalakshmi, T. R.; AravindhaBabu, N.; Shanmugam, K. T.; Masthan, K. M. K.

    2015-01-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  1. Speciation of oxaliplatin adducts with DNA nucleotides.

    PubMed

    Zayed, Aref; Jones, George D D; Reid, Helen J; Shoeib, Tamer; Taylor, Sarah E; Thomas, Anne L; Wood, Joanna P; Sharp, Barry L

    2011-10-01

    This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy.

  2. DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas. Potential use for genotoxicant biomonitoring of fresh water ecosystems.

    PubMed

    Le Goff, J; Gallois, J; Pelhuet, L; Devier, M H; Budzinski, H; Pottier, D; André, V; Cachot, J

    2006-08-12

    The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 microg g(-1) dry weight) in comparison to individuals from the reference site (0.053 microg g(-1) dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10(8) nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 microg g(-1) dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 microg g(-1) dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10(8) nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable

  3. Quantitative changes in endogenous DNA adducts correlate with conazole in vivo mutagenicity and tumorigenicity.

    PubMed

    Ross, Jeffrey A; Leavitt, Sharon A; Schmid, Judith E; Nelson, Garret B

    2012-09-01

    The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by (32)P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups (P = 0.002), as were total endogenous DNA adduct levels (P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with

  4. Imidazolidinone adducts of peptides and hemoglobin

    SciTech Connect

    San George, R.C.; Hoberman, H.D.

    1986-05-01

    Acetaldehyde reacts selectively with the terminal amino groups of the ..cap alpha.. and ..beta.. chains of hemoglobin to form stable adducts, the structures of which, based on /sup 13/C NMR studies, are proposed to be diastereomeric 2-methyl imidazolidin-4-ones. In this scheme, acetaldelhyde forms a reversible Schiff base with the ..cap alpha..-amino groups of the polypeptide chains which cyclize with the amide nitrogen of the first peptide bond to form the stable imidazolidinone adducts. In support of this mechanism, the authors found that in following the reaction of the peptide val-gly-gly with (1,2-/sup 13/C) acetaldehyde, /sup 13/C NMR resonances attributed to a Schiff base (delta = 170 ppm) were observed which slowly disappeared prior to appearance of resonances from a pair of stable adducts (delta = 70 and 71 ppm) believed to be the diastereomeric imidazolidinones. Schiff base formation appeared to limit the overall rate. Tetraglycine reacted in a similar manner but with a resonance from a single stable adduct observed representing the enantiomeric imidazolidinone adducts of this peptide. Peptides with proline in position 2 should be incapable of forming imidazolidinones, and the authors found that ala-pro-gly did in fact fail to form a stable adduct with acetaldehyde. The 2-methyl imidazolidin-4-one adducts of hemoglobin may be useful in determining the contribution of the amino terminal groups to the structure and functional properties of hemoglobins.

  5. [DNA adducts in human female genital organs].

    PubMed

    Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Monist, Marta; Baranowski, Włodzimierz

    2007-12-01

    DNA adducts, one of genetic damages markers, precede and finally can lead to oncogenic mutations. They appear in genome as a result of DNA bases damages caused by various and numerous environmental factors eg. ultraviolet light, ionic radiation, toxins and also endogenic substances, for example estrogens. It is believed that the creation of DNA adducts is a necessary but insufficient process for the neoplastic transformation of the cell. The following review presents concise knowledge about the DNA adducts creation and their sequels served in healthy and cancerous tissues of the female genital organs, on the base of the available data. PMID:18411923

  6. Detection of cellular DNA adducts in human fibroblasts treated with A-ring saturated and A-NCR-DMBA

    SciTech Connect

    Kumari, H.L.; Abood, N.; Goswami, S.P.; Bhat, H.B.; Milo, G.E.; Vitiak, D.T.

    1986-05-01

    Previous reports from these laboratories revealed that 1,2,3,4-tetrahydro 7,12-dimethylbenz(a)anthracene (TH-DMBA) and 6,11-dimethylcyclopentano(a)anthracene (CP-DMA) transform human fibroblasts to an abnormal phenotype. The parent PAH 7,12-DMBA does not transform these cells. Since 7,12-DMBA but not TH-DMBA or CP-DMA is anticipated to form active bay region diol epoxides, they investigated whether the A-ring analogues form adducts with cellular DNA, when the cells are treated in S phase of the cell cycle. Treatment for 10 hr was carried out when the cells exhibited S phase entry i.e. 10 hr after Gl release from a nutritionally deficient block. Adducts were isolated using a modification of the /sup 32/P-post-labeling technique. Preliminary results indicate that TH-DMBA formed two different nucleotide adducts where as there were three different adducts identified with CP-DMA. These results suggest the level of modification to be circa 1-2 adducts/10/sup 7/ total nucleotides for TH-DMBA and 2-4 adducts/10/sup 7/ total nucleotides for CP-DMA. The present evidence accumulated to date strongly suggests that alternate mechanisms exist to oxygenate the FAH to reactive intermediates that subsequently form specific DNA adducts.

  7. Sperm DNA oxidative damage and DNA adducts.

    PubMed

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

    2015-12-01

    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 sperm.

  8. Sperm DNA oxidative damage and DNA adducts.

    PubMed

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

    2015-12-01

    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 sperm

  9. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  10. 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

  11. Macromolecular adducts caused by environmental chemicals.

    PubMed

    Neumann, H G; Albrecht, O; van Dorp, C; Zwirner-Baier, I

    1995-12-01

    We describe three biomonitoring studies in which hemoglobin (Hb) adducts were used as biochemical markers to assess indirectly the target dose of genotoxic chemicals. We monitored the exposure to 1,3-butadiene in occupationally exposed workers and in two control groups by analyzing the adducts formed by the reaction of the first activation product, butadiene monoepoxide, with the terminal valine of Hb; we also measured hydrolyzable adducts formed by the reaction of metabolically formed nitroso derivatives with Hb from five selected nitropolycyclic aromatic hydrocarbons (1-nitropyrene; 2-nitrofluorene, 3-nitrofluoranthrene, 6-nitrochrysene, and 9-nitrophenanthrene) in coke oven workers of different job categories and control workers of the same geographical area. We detected hydrolyzable adducts from monocyclic nitroarenes in blood from individuals living in a contaminated area where explosives had been produced and from controls. The contaminants considered were 2,4,6-trinitrotoluene; 2,4- and 2,6-dinitrotoluene; and 1,3-dinitrobenzene. Differences between groups were significant, but interindividual variation was great and back-ground exposures must be considered.

  12. Adducts of Oxylipin Electrophiles to Glutathione Reflect a 13 Specificity of the Downstream Lipoxygenase Pathway in the Tobacco Hypersensitive Response

    PubMed Central

    Davoine, Céline; Falletti, Olivier; Douki, Thierry; Iacazio, Gilles; Ennar, Najla; Montillet, Jean-Luc; Triantaphylidès, Christian

    2006-01-01

    The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR. PMID:16500992

  13. Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms.

    PubMed

    Yang, Xiangkun; Bartlett, Michael G

    2016-03-15

    The determination of protein-xenobiotic adducts using mass spectrometry is an emerging area which allows detailed understanding of the underlying mechanisms involved in toxicity. These approaches can also be used to reveal potential biomarkers of exposure or toxic response. The following review covers studies of protein adducts resulting from exposure to a wide variety of xenobiotics including organophosphates, polycyclic aromatic hydrocarbons, acetaminophen, alkylating agents and other related compounds. PMID:26842586

  14. Antitumour 2-(4-aminophenyl)benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo

    PubMed Central

    Leong, C-O; Gaskell, M; Martin, E A; Heydon, R T; Farmer, P B; Bibby, M C; Cooper, P A; Double, J A; Bradshaw, T D; Stevens, M F G

    2003-01-01

    2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 μM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 μM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 μM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) ≥100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 μM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 μM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (⩽10 μM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg−1). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse. PMID:12569393

  15. PARP1 impact on DNA repair of platinum adducts: preclinical and clinical read-outs.

    PubMed

    Olaussen, Ken A; Adam, Julien; Vanhecke, Elsa; Vielh, Philippe; Pirker, Robert; Friboulet, Luc; Popper, Helmut; Robin, Angélique; Commo, Fréderic; Thomale, Jürgen; Kayitalire, Louis; Filipits, Martin; Le Chevalier, Thierry; André, Fabrice; Brambilla, Elisabeth; Soria, Jean-Charles

    2013-05-01

    Evaluation of DNA repair proteins might provide meaningful information in relation to prognosis and chemotherapy efficacy in Non-Small Cell Lung Cancer (NSCLC) patients. The role of Poly(ADP-Ribose) Polymerase (PARP) in DNA repair of platinum adducts has not been firmly established. We used a DNA repair functional test based on antibody recognition of cisplatin intrastrand platinum adducts on DNA. We evaluated the effect of PARP inhibition on DNA repair functionality in a panel of cisplatin cell lines treated by the clinical-grade pharmacological inhibitor CEP8983 (a 4-methoxy-carbazole derivate) and the commercially available inhibitor PJ34 (phenanthridinone). We determined PARP1 protein expression in whole tumor sections from the International Adjuvant Lung cancer Trial (IALT)-bio study and tested a 3-marker PARP1/MSH2/ERCC1 algorithm combining PARP1 tumor status with previously published data. Chemosensitivity of cisplatin in NSCLC cell lines was correlated with the accumulation of cisplatin DNA adducts (P=0.0004). Further, the pharmacological inhibition of PARP induced a 1.7 to 2.3-fold increase in platinum adduct accumulation (24h) in A549 cell line suggesting a slow-down of platinum DNA-adduct repair capacity. In parallel, PARP1 inhibition increased the sensitivity to cisplatin treatment. In patient samples, PARP1 expression levels did not influence patient survival or the effect of platinum-based post-operative chemotherapy in the global IALT-bio population (interaction P=0.79). Among cases with high expression of all three markers (triple positive), untreated patients had prolonged survival with a median DFS of 7.8 years, (HR=0.34, 95%CI [0.19-0.61], adjusted P=0.0003) compared to triple negative patients (1.4 years). Remarkably, triple positive patients suffered from a detrimental effect (4.9-year reduction of median DFS) by post-operative cisplatin-based chemotherapy (HR=1.79, 95%CI [1.01-3.17], adjusted P=0.04, chemotherapy vs. control). Combinatorial

  16. Control of DNA hybridization with photocleavable adducts.

    PubMed

    Ghosn, Bilal; Haselton, Frederick R; Gee, Kyle R; Monroe, W Todd

    2005-01-01

    Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity.

  17. Inhibition by resistant starch of red meat-induced promutagenic adducts in mouse colon.

    PubMed

    Winter, Jean; Nyskohus, Laura; Young, Graeme P; Hu, Ying; Conlon, Michael A; Bird, Anthony R; Topping, David L; Le Leu, Richard K

    2011-11-01

    Population studies have shown that high red meat intake may increase colorectal cancer risk. Our aim was to examine the effect of different amounts and sources of dietary protein on induction of the promutagenic adduct O(6)-methyl-2-deoxyguanosine (O(6)MeG) in colonocytes, to relate these to markers of large bowel protein fermentation and ascertain whether increasing colonic carbohydrate fermentation modified these effects. Mice (n = 72) were fed 15% or 30% protein as casein or red meat or 30% protein with 10% high amylose maize starch as the source of resistant starch. Genetic damage in distal colonocytes was detected by immunohistochemical staining for O(6)MeG and apoptosis. Feces were collected for measurement of pH, ammonia, phenols, p-cresol, and short-chain fatty acids (SCFA). O(6)MeG and fecal p-cresol concentrations were significantly higher with red meat than with casein (P < 0.018), with adducts accumulating in cells at the crypt apex. DNA adducts (P < 0.01) and apoptosis (P < 0.001) were lower and protein fermentation products (fecal ammonia, P < 0.05; phenol, P < 0.0001) higher in mice fed resistant starch. Fecal SCFA levels were also higher in mice fed resistant starch (P < 0.0001). This is the first demonstration that high protein diets increase promutagenic adducts (O(6)MeG) in the colon and dietary protein type seems to be the critical factor. The delivery of fermentable carbohydrate to the colon (as resistant starch) seems to switch from fermentation of protein to that of carbohydrate and a reduction in adduct formation, supporting previous observations that dietary resistant starch opposes the mutagenic effects of dietary red meat.

  18. Human DNA adduct measurements: state of the art.

    PubMed Central

    Poirier, M C; Weston, A

    1996-01-01

    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 32P-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. PMID:8933030

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. Accumulate-Repeat-Accumulate-Accumulate-Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Sam; Thorpe, Jeremy

    2004-01-01

    Inspired by recently proposed Accumulate-Repeat-Accumulate (ARA) codes [15], in this paper we propose a channel coding scheme called Accumulate-Repeat-Accumulate-Accumulate (ARAA) codes. These codes can be seen as serial turbo-like codes or as a subclass of Low Density Parity Check (LDPC) codes, and they have a projected graph or protograph representation; this allows for a high-speed iterative decoder implementation using belief propagation. An ARAA code can be viewed as a precoded Repeat-and-Accumulate (RA) code with puncturing in concatenation with another accumulator, where simply an accumulator is chosen as the precoder; thus ARAA codes have a very fast encoder structure. Using density evolution on their associated protographs, we find examples of rate-lJ2 ARAA codes with maximum variable node degree 4 for which a minimum bit-SNR as low as 0.21 dB from the channel capacity limit can be achieved as the block size goes to infinity. Such a low threshold cannot be achieved by RA or Irregular RA (IRA) or unstructured irregular LDPC codes with the same constraint on the maximum variable node degree. Furthermore by puncturing the accumulators we can construct families of higher rate ARAA codes with thresholds that stay close to their respective channel capacity thresholds uniformly. Iterative decoding simulation results show comparable performance with the best-known LDPC codes but with very low error floor even at moderate block sizes.

  4. Accumulate-Repeat-Accumulate-Accumulate Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Samuel; Thorpe, Jeremy

    2007-01-01

    Accumulate-repeat-accumulate-accumulate (ARAA) codes have been proposed, inspired by the recently proposed accumulate-repeat-accumulate (ARA) codes. These are error-correcting codes suitable for use in a variety of wireless data-communication systems that include noisy channels. ARAA codes can be regarded as serial turbolike codes or as a subclass of low-density parity-check (LDPC) codes, and, like ARA codes they have projected graph or protograph representations; these characteristics make it possible to design high-speed iterative decoders that utilize belief-propagation algorithms. The objective in proposing ARAA codes as a subclass of ARA codes was to enhance the error-floor performance of ARA codes while maintaining simple encoding structures and low maximum variable node degree.

  5. 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...

  6. 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. PMID:21856030

  7. Fluorescence of Phytochrome Adducts with Synthetic Locked Chromophores*

    PubMed Central

    Zienicke, Benjamin; Chen, Li-Yi; Khawn, Htoi; Hammam, Mostafa A. S.; Kinoshita, Hideki; Reichert, Johannes; Ulrich, Anne S.; Inomata, Katsuhiko; Lamparter, Tilman

    2011-01-01

    We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion. PMID:21071442

  8. Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation.

    PubMed

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Chin, Chiu; Huang, William; Lepor, Herbert; Wu, Xue-Ru; Rom, William N; Chen, Lung-Chi; Tang, Moon-shong

    2015-10-20

    Second-hand smoke (SHS) is associated with 20-30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers. PMID:26431382

  9. Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation.

    PubMed

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Chin, Chiu; Huang, William; Lepor, Herbert; Wu, Xue-Ru; Rom, William N; Chen, Lung-Chi; Tang, Moon-shong

    2015-10-20

    Second-hand smoke (SHS) is associated with 20-30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers.

  10. Role of pyridine in Wyodak-pyridine adducts

    SciTech Connect

    David L. Wertz; Amanda Winters; Tara Craft; Jami Holloway

    2006-02-01

    When pyridine (PYR) is added to powdered Wyodak subbituminous coal (WYO), the sample is converted to a paste, and the molecular-level adduct which is formed is stable for months. After the excess pyridine has evaporated from the WYO-PYR sample, the stoichiometry of the adduct is ca. two pyridine molecules per bilayer of WYO polycyclic units; this adduct exists even after mild vacuum treatment of the sample. The pyridine molecules in this adduct appear to be located between the bilayer lamellae and to be H-bonded to either H-O or H-N moieties attached to the poly-cyclic aromatic units of WYO. An H-bonded N- - -H-X distance of 2.6 {angstrom} has been calculated from a structural model of the WYO-PYR adduct. 37 refs., 12 figs., 4 tabs.

  11. General method for quantifying base adducts in specific mammalian genes.

    PubMed Central

    Thomas, D C; Morton, A G; Bohr, V A; Sancar, A

    1988-01-01

    A general method has been developed to measure the formation and removal of DNA adducts in defined sequences of mammalian genomes. Adducted genomic DNA is digested with an appropriate restriction enzyme, treated with Escherichia coli UvrABC excision nuclease (ABC excinuclease), subjected to alkaline gel electrophoresis, and probed for specific sequences by Southern hybridization. The ABC excinuclease incises DNA containing bulky adducts and thus reduces the intensity of the full-length fragments in Southern hybridization in proportion to the number of adducts present in the probed sequence. This method is similar to that developed by Bohr et al. [Bohr, V. A., Smith, C. A., Okumoto, D. S. & Hanawalt, P. C. (1985) Cell 40, 359-369] for quantifying pyrimidine dimers by using T4 endonuclease V. Because of the wide substrate range of ABC exinuclease, however, our method can be used to quantify a large variety of DNA adducts in specific genomic sequences. Images PMID:2836856

  12. The knee adduction moment during gait is associated with the adduction angle measured during computer-assisted total knee arthroplasty.

    PubMed

    Roda, Richard D; Wilson, Janie L Astephen; Wilson, David A J; Richardson, Glen; Dunbar, Michael J

    2012-06-01

    Computer-assisted surgery can be used to measure 3-dimensional knee function during arthroplasty surgery; however, it is unknown if the movement of the knee measured during surgery is related to the in vitro, dynamic state of the knee joint, specifically the knee adduction moment during gait, which has been related to implant migration. The purpose of this study was to determine if the preoperative adduction moment is correlated with the knee abduction/adduction angle measured intraoperatively. A statistically significant correlation was found between the mean (r(2) = 0.59; P = .001) and peak (r(2) = 0.53; P = .003) preoperative knee adduction moment and the mean abduction/adduction angle measured intraoperatively. The association found in this study suggests the potential for incorporating functional information that relates to surgical outcome into surgical decision making using computer-assisted surgery.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Probing lipid-protein adduction with alkynyl surrogates: application to Smith-Lemli-Opitz syndrome[S

    PubMed Central

    Windsor, Katherine; Genaro-Mattos, Thiago C.; Kim, Hye-Young H.; Liu, Wei; Tallman, Keri A.; Miyamoto, Sayuri; Korade, Zeljka; Porter, Ned A.

    2013-01-01

    Lipid modifications aid in regulating (and misregulating) protein function and localization. However, efficient methods to screen for a lipid's ability to modify proteins are not readily available. We present a strategy to identify protein-reactive lipids and apply it to a neurodevelopmental disorder, Smith-Lemli-Opitz syndrome (SLOS). Alkynyl surrogates were synthesized for polyunsaturated fatty acids, phospholipids, cholesterol, 7-dehydrocholesterol (7-DHC), and a 7-DHC-derived oxysterol. To probe for protein-reactive lipids, we used click chemistry to biotinylate the alkynyl tag and detected the lipid-adducted proteins with streptavidin Western blotting. In Neuro2a cells, the trend in amount of protein adduction followed known rates of lipid peroxidation (7-DHC >> arachidonic acid > linoleic acid >> cholesterol), with alkynyl-7-DHC producing the most adduction among alkynyl lipids. 7-DHC reductase-deficient cells, which cannot properly metabolize 7-DHC, exhibited significantly more alkynyl-7-DHC-protein adduction than control cells. Model studies demonstrated that a 7-DHC peroxidation product covalently modifies proteins. We hypothesize that 7-DHC generates electrophiles that can modify the proteome, contributing to SLOS's complex pathology. These probes and methods would allow for analysis of lipid-modified proteomes in SLOS and other disorders exhibiting 7-DHC accumulation. More broadly, the alkynyl lipid library would facilitate exploration of lipid peroxidation's role in specific biological processes in numerous diseases. PMID:23828810

  19. In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide adducts.

    PubMed

    Chary, P; Lloyd, R S

    1995-04-25

    DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in approximately 16% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3'-->5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.

  20. Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases.

    PubMed

    Pham, Robert T; Gardner, James L; Gallagher, Evan P

    2002-01-01

    The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.

  1. Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner.

    PubMed

    Riahi, Yael; Kaiser, Nurit; Cohen, Guy; Abd-Elrahman, Ihab; Blum, Galia; Shapira, Oz M; Koler, Tomer; Simionescu, Maya; Sima, Anca V; Zarkovic, Neven; Zarkovic, Kamelija; Orioli, Marica; Aldini, Giancarlo; Cerasi, Erol; Leibowitz, Gil; Sasson, Shlomo

    2015-08-01

    Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.

  2. The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal.

    PubMed

    Forman, Henry Jay; Fukuto, Jon M; Miller, Tom; Zhang, Hongqiao; Rinna, Alessandra; Levy, Smadar

    2008-09-15

    During the past several years, major advances have been made in understanding how reactive oxygen species (ROS) and nitrogen species (RNS) participate in signal transduction. Identification of the specific targets and the chemical reactions involved still remains to be resolved with many of the signaling pathways in which the involvement of reactive species has been determined. Our understanding is that ROS and RNS have second messenger roles. While cysteine residues in the thiolate (ionized) form found in several classes of signaling proteins can be specific targets for reaction with H(2)O(2) and RNS, better understanding of the chemistry, particularly kinetics, suggests that for many signaling events in which ROS and RNS participate, enzymatic catalysis is more likely to be involved than non-enzymatic reaction. Due to increased interest in how oxidation products, particularly lipid peroxidation products, also are involved with signaling, a review of signaling by 4-hydroxy-2-nonenal (HNE) is included. This article focuses on the chemistry of signaling by ROS, RNS, and HNE and will describe reactions with selected target proteins as representatives of the mechanisms rather attempt to comprehensively review the many signaling pathways in which the reactive species are involved.

  3. Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner

    PubMed Central

    Riahi, Yael; Kaiser, Nurit; Cohen, Guy; Abd-Elrahman, Ihab; Blum, Galia; Shapira, Oz M; Koler, Tomer; Simionescu, Maya; Sima, Anca V; Zarkovic, Neven; Zarkovic, Kamelija; Orioli, Marica; Aldini, Giancarlo; Cerasi, Erol; Leibowitz, Gil; Sasson, Shlomo

    2015-01-01

    Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence. PMID:25754218

  4. DNA and protein adducts as markers of genotoxicity.

    PubMed

    Farmer, Peter B

    2004-04-01

    Determination of the interaction products (adducts) of a carcinogen with DNA or protein indicates the amount of genotoxic material that has reached the tissue under study and provides a valuable biomarker of exposure for molecular epidemiological studies. DNA adducts may also give further information with regard to the mutagenic significance of the exposure. The sensitivity and applicability of the analytical methods for the detection and quantification of carcinogen adducts has greatly increased in recent years, and DNA damage levels as low as one adduct per 10(8) nucleotides can now routinely be measured. The discovery of many types of endogenously-produced damage of DNA and protein has demonstrated previously unsuspected sources of genotoxicity, the biological consequences of which are so far not known.

  5. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    DOE PAGES

    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 themore » first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.« less

  6. 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.

  7. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity.

    PubMed

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

    2015-12-21

    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 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) 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.

  8. Selective syntheses of novel polyether fullerene multiple adducts.

    PubMed

    Zhou, Zhiguo; Schuster, David I; Wilson, Stephen R

    2003-10-01

    We have applied a modified macrocyclic tether approach to control multiple additions to C60. The technique of 3He NMR was used to confirm the selective formation of specific C60 multiple adducts by the macrocyclic tether approach. An oligoglycol was used as a flexible linker to produce macrocyclic polyether-linked malonates 5, 6, 8, and 9 under solid-liquid PTC (phase-transfer-catalysis) conditions. The formation of a single C60 tris-adduct, 3, from macrocyclic malonate 1 and 3He@C60 was proven by 3He NMR. Similarly, multiple additions to C60 of macrocyclic polyether malonate 5 gave C60 bis-adduct 10 selectively, while the reaction of C60 with macrocyclic malonate 8 gave bis-adducts 11 and 12. A similar process with macrocyclic malonate 6 gave tris-adduct 13 with high selectivity as well. Saponification of these C60 multiple adducts gives the corresponding polyacids that are potentially useful in biological applications. Macrocyclic polyether fullerenes are a new class of ionophores, which could be interesting for molecular recognition and for the development of biosensors.

  9. Relating protein adduction to gene expression changes: a systems approach

    PubMed Central

    Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

    2013-01-01

    Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data. PMID:21594272

  10. ³²P-postlabeling analysis of DNA adducts.

    PubMed

    Phillips, David H; Arlt, Volker M

    2014-01-01

    (32)P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of (32)P-orthophosphate from [γ-(32)P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10(9)-10(10) normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair. PMID:24623224

  11. Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells.

    PubMed

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Hu, Yu; Chen, Wei-sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A; Tang, Moon-shong

    2014-06-15

    Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.

  12. Stability of acetaldehyde-derived DNA adduct in vitro.

    PubMed

    Hori, Kimiko; Miyamoto, Shin'ichi; Yukawa, Yoshiyuki; Muto, Manabu; Chiba, Tsutomu; Matsuda, Tomonari

    2012-07-13

    Acetaldehyde (AA) derived from alcoholic beverages is a confirmed carcinogen for esophageal and head and neck cancers. AA forms various DNA adducts and is thought to play a crucial role in carcinogenesis. Transient DNA adducts are usually repaired, but the stability of AA-derived DNA adducts has not been elucidated. We investigated the stability of N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG), a major AA-derived DNA adduct, in cultured cells. First, to determine the optimal concentration of AA for detecting N(2)-ethylidene-dG in cell culture, a dose-response study was performed using HL60 cells of the human promyelocytic leukemia cell line. An AA concentration ≥ 0.01% (1.8 mM) was required to detect N(2)-ethylidene-dG in vitro. We next examined the stability of N(2)-ethylidene-dG. After a 1 or 2h exposure to 0.01% of AA in a tightly sealed bottle, N(2)-ethylidene-dG content was measured by sensitive liquid chromatography tandem mass spectrometry immediately, 24h, and 48 h after exposure. After the 1h exposure, the mean (± SD) N(2)-ethylidene-dG contents were 12.1 ± 1.28, 8.20 ± 0.64, and 6.70 ± 0.52 adducts per 10(7) bases at each postexposure time. After the 2h exposure, N(2)-ethylidene-dG content increased to 21.4 ± 7.50, 10.5 ± 3.61, and 9.83 ± 3.90 adducts per 10(7) bases at each postexposure time. The half-life of this adduct was calculated as ∼35 h in independent experiments. These results indicate that AA exposure from daily alcohol consumption may cause DNA damage and may increase the risk of alcohol-related carcinogenesis. PMID:22683642

  13. 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

    2014-05-01

    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.

  14. 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. PMID:26291948

  15. Structures of oxaliplatin-oligonucleotide adducts from DNA.

    PubMed

    Mowaka, Shereen; Ziehe, Matthias; Mohamed, Dalia; Hochkirch, Ulrike; Thomale, Jürgen; Linscheid, Michael W

    2012-10-01

    Oxaliplatin, [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II) shows a great efficiency against colorectal cancer. Although the mode of action of oxaliplatin is not yet understood, it is commonly accepted that binding of oxaliplatin to DNA prevents DNA synthesis and alters protein to DNA binding. In order to elucidate the modified DNA-protein interaction and thus to understand the mechanisms leading to cellular misinterpretation of DNA information and apoptosis, we have identified the preferential binding sites and the dynamics of the oxaliplatin-DNA intrastrand and interstrand adducts at the oligomer level using high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) and HPLC/inductively coupled plasma-MS for quantitative studies. We used a combination of benzonase, alkaline phosphatase and Nuclease S1 for digestion. This digestion procedure allows the study of platinated oligomeric nucleotides and more complex interstrand adducts. The digestion products were mostly chromatographically separated and characterized using HPLC/ESI-ion trap MS/MS experiments. We could show that the adducts to guanine and adenine are quite dynamic; that is, the ratios are changing for several days. In addition, the resulting adducts provide evidence for the action of the digesting enzymes and indicate that the adduct spectrum at the oligomeric level is different to that at the commonly studies dinucleotide level.

  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. 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.

  18. 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

  19. Accumulate Repeat Accumulate Coded Modulation

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative coded modulation scheme called 'Accumulate Repeat Accumulate Coded Modulation' (ARA coded modulation). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes that are combined with high level modulation. Thus at the decoder belief propagation can be used for iterative decoding of ARA coded modulation on a graph, provided a demapper transforms the received in-phase and quadrature samples to reliability of the bits.

  20. 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.

  1. 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.

  2. Recognition of platinum-DNA adducts by HMGB1a.

    PubMed

    Ramachandran, Srinivas; Temple, Brenda; Alexandrova, Anastassia N; Chaney, Stephen G; Dokholyan, Nikolay V

    2012-09-25

    Cisplatin (CP) and oxaliplatin (OX), platinum-based drugs used widely in chemotherapy, form adducts on intrastrand guanines (5'GG) in genomic DNA. DNA damage recognition proteins, transcription factors, mismatch repair proteins, and DNA polymerases discriminate between CP- and OX-GG DNA adducts, which could partly account for differences in the efficacy, toxicity, and mutagenicity of CP and OX. In addition, differential recognition of CP- and OX-GG adducts is highly dependent on the sequence context of the Pt-GG adduct. In particular, DNA binding protein domain HMGB1a binds to CP-GG DNA adducts with up to 53-fold greater affinity than to OX-GG adducts in the TGGA sequence context but shows much smaller differences in binding in the AGGC or TGGT sequence contexts. Here, simulations of the HMGB1a-Pt-DNA complex in the three sequence contexts revealed a higher number of interface contacts for the CP-DNA complex in the TGGA sequence context than in the OX-DNA complex. However, the number of interface contacts was similar in the TGGT and AGGC sequence contexts. The higher number of interface contacts in the CP-TGGA sequence context corresponded to a larger roll of the Pt-GG base pair step. Furthermore, geometric analysis of stacking of phenylalanine 37 in HMGB1a (Phe37) with the platinated guanines revealed more favorable stacking modes correlated with a larger roll of the Pt-GG base pair step in the TGGA sequence context. These data are consistent with our previous molecular dynamics simulations showing that the CP-TGGA complex was able to sample larger roll angles than the OX-TGGA complex or either CP- or OX-DNA complexes in the AGGC or TGGT sequences. We infer that the high binding affinity of HMGB1a for CP-TGGA is due to the greater flexibility of CP-TGGA compared to OX-TGGA and other Pt-DNA adducts. This increased flexibility is reflected in the ability of CP-TGGA to sample larger roll angles, which allows for a higher number of interface contacts between the Pt

  3. Characterization of oxaliplatin-DNA adduct formation in DNA and differentiation of cancer cell drug sensitivity at microdose concentrations.

    PubMed

    Hah, Sang Soo; Sumbad, Rhoda A; de Vere White, Ralph W; Turteltaub, Kenneth W; Henderson, Paul T

    2007-12-01

    (trans-R, R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a recently approved platinum analogue for use in the chemotherapy of metastatic colorectal cancer. Like many cytotoxic drugs, oxaliplatin exerts its antitumor effects by covalent modification of DNA. We report an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair. We determined the apparent rate of oxaliplatin adduction to salmon sperm DNA. The oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by HPLC-AMS. Cultured platinum-sensitive testicular (833K) and platinum-resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells were incubated with a subpharmacological concentration of oxaliplatin (0.2 microM). Both cellular and DNA radiocarbon contents in the drug-sensitive testicular cells had approximately twice the area under the curve as compared to the more platinum-resistant cell lines, implying that differential accumulation of the drug may be responsible for the sensitivity of cancer cells to platinum treatment. The lowest concentration of radiocarbon measured was approximately 1+/-0.1 amol/microg of DNA, when assaying 1 microg of DNA. This sensitivity for measuring oxaliplatin-DNA adducts is the highest reported to date. The sensitivity offered by this method may be applicable to other DNA-damaging drugs, metabolisms studies, and diagnostics development.

  4. N-Heterocyclic carbene phosphaketene adducts as precursors to carbene-phosphinidene adducts and a rearranged π-system.

    PubMed

    Li, Zhongshu; Chen, Xiaodan; Li, Yaqi; Su, Cheng-Yong; Grützmacher, Hansjörg

    2016-10-15

    The nucleophilic attack of NHCs on the electron deficient carbon of tetryl substituted phosphaketenes Ph3E-P[double bond, length as m-dash]C[double bond, length as m-dash]O (E = Sn-Si), leads quantitatively to the formation of NHC-phosphaketene adducts. With E = Sn or Ge, these zwitterionic adducts decompose upon thermolysis under the release of carbon monoxide to give zwitterionic NHC-phosphinidene adducts. With E = Si an OCP to CPO rearrangement occurs which leads to the formation of a linear π-conjugated molecule, NHC[double bond, length as m-dash]C[double bond, length as m-dash]P-O-SiPh3. PMID:27545980

  5. 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. PMID:27046699

  6. 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. PMID:27180422

  7. 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.

  8. Heat accumulator

    SciTech Connect

    Bracht, A.

    1981-09-29

    A heat accumulator comprises a thermally-insulated reservoir full of paraffin wax mixture or other flowable or meltable heat storage mass, heat-exchangers immersed in the mass, a heat-trap connected to one of the heat-exchangers, and a heat user connected to the other heat-exchanger. Pumps circulate fluids through the heat-trap and the heat-using means and the respective heat-exchangers, and a stirrer agitates and circulates the mass, and the pumps and the stirrer and electric motors driving these devices are all immersed in the mass.

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 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...

  11. 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...

  12. 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)...

  13. 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)...

  14. 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)...

  15. 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)...

  16. Adduct formation and repair, and translesion DNA synthesis across the adducts in human cells exposed to 3-nitrobenzanthrone.

    PubMed

    Kawanishi, Masanobu; Fujikawa, Yoshihiro; Ishii, Hiroshi; Nishida, Hiroshi; Higashigaki, Yuka; Kanno, Takaharu; Matsuda, Tomonari; Takamura-Enya, Takeji; Yagi, Takashi

    2013-05-15

    3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a potent environmental mutagen that is found in diesel exhaust fumes and airborne particulates. It is known to produce several DNA adducts, including three major adducts N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-ABA), 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone (dA-N(6)-C2-ABA), and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-C2-ABA) in mammalian cells. In the present study, we measured the quantity of the formation and subsequent reduction of these adducts in human hepatoma HepG2 cells that had been treated with 3-NBA using LC-MS/MS analysis. As a result, dG-C8-N-ABA and dG-N(2)-C2-ABA were identified as major adducts in the HepG2 cells, and dA-N(6)-C2-ABA was found to be a minor adduct. Treatment with 1μg/mL 3-NBA for 24h induced the formation of 2835±1509 dG-C8-N-ABA and 3373±1173 dG-N(2)-C2-ABA per 10(7) dG and 877±330 dA-N(6)-C2-ABA per 10(7) dA in the cells. The cellular DNA repair system removed the dG-C8-N-ABA and dA-N(6)-C2-ABA adducts more efficiently than the dG-N(2)-C2-ABA adducts. After a 24-h repair period, 86.4±11.1% of the dG-N(2)-C2-ABA adducts remained, whereas only 51.7±2.7% of the dG-C8-N-ABA adducts and 37.8±1.7% of the dA-N(6)-C2-ABA adducts were present in the cells. We also evaluated the efficiency of bypasses across these three adducts and their mutagenic potency by introducing site-specific mono-modified plasmids into human cells. This translesion DNA synthesis (TLS) assay showed that dG-C8-N-ABA blocked DNA replication markedly (its replication frequency was 16.9±2.7%), while the replication arrests induced by dG-N(2)-C2-ABA and dA-N(6)-C2-ABA were more moderate (their replication frequencies were 33.3±6.2% and 43.1±7.5%, respectively). Mutagenic TLS was observed more frequently in replication across dG-C8-N-ABA (30.6%) than in replication across dG-N(2)-C2-ABA (12.1%) or dA-N(6)-C2-ABA (12.1%). These findings provide important

  17. 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.

  18. Mass Spectrometric Analyses of Organophosphate Insecticide Oxon Protein Adducts

    PubMed Central

    Thompson, Charles M.; Prins, John M.; George, Kathleen M.

    2010-01-01

    Objective Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. Data sources and extraction We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. Data synthesis A number of OP-based insecticides share common structural elements that result in predictable OP–protein adducts. The resultant OP–protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. Conclusions MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure. PMID:20056576

  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. PMID:26700446

  20. 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.

  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. Towards biomarker-dependent individualized chemotherapy: exploring cell-specific differences in oxaliplatin-DNA adduct distribution using accelerator mass spectrometry.

    PubMed

    Hah, Sang Soo; Henderson, Paul T; Turteltaub, Kenneth W

    2010-04-15

    Oxaliplatin is a third-generation platinum-based anticancer drug that is currently used in the treatment of metastatic colorectal cancer. Oxaliplatin, like other platinum-based anticancer drugs such as cisplatin and carboplatin, is known to induce apoptosis in tumor cells by binding to nuclear DNA, forming monoadducts, and intra- and interstrand diadducts. Previously, we reported an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair [Hah, S. S.; Sumbad, R. A.; de Vere White, R. W.; Turteltaub, K. W.; Henderson, P. T. Chem. Res. Toxicol.2007, 20, 1745]. Here, we describe another application of AMS to the measurement of oxaliplatin-DNA adduct distribution in cultured platinum-sensitive testicular (833K) and platinum-resistant breast (MDA-MB-231) cancer cells, which resulted in elucidation of cell-dependent differentiation of oxaliplatin-DNA adduct formation, implying that differential adduction and/or accumulation of the drug in cellular DNA may be responsible for the sensitivity of cancer cells to platinum treatment. Ultimately, we hope to use this method to measure the intrinsic platinated DNA adduct repair capacity in cancer patients for use as a biomarker for diagnostics or a predictor of patient outcome.

  3. 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.

  4. 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.

  5. Human Biomonitoring of DNA Adducts by Ion Trap Multistage Mass Spectrometry.

    PubMed

    Guo, Jingshu; Turesky, Robert J

    2016-01-01

    Humans are continuously exposed to hazardous chemicals in the environment. These chemicals or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. The identification of DNA adducts is required for understanding exposure and the etiological role of a genotoxic chemical in cancer risk. The analytical chemist is confronted with a great challenge because the levels of DNA adducts generally occur at <1 adduct per 10(7) nucleotides, and the amount of tissue available for measurement is limited. Ion trap mass spectrometry has emerged as an important technique to screen for DNA adducts because of the high level sensitivity and selectivity, particularly when employing multi-stage scanning (MS(n) ). The product ion spectra provide rich structural information and corroborate the adduct identities even at trace levels in human tissues. Ion trap technology represents a significant advance in measuring DNA adducts in humans. © 2016 by John Wiley & Sons, Inc. PMID:27584705

  6. 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.

  7. 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.

  8. Dispersant additives derived from lactone modified amido-amine adducts

    SciTech Connect

    Gutierrez, A.; Lundberg, R.D.

    1990-10-16

    This patent describes a lactone modified dispersant additive. It comprises one adduct of a polyolefin of 300 to 10,000 number average molecular weight substituted with at least 0.8 (e.g., from about 1 to 4) dicarboxylic acid producing moieties (preferably acid or anhydride moieties) per polyolefin molecule, an amido-amine or thioamido-amine characterized by being a reaction product of at least a polyamine and an alpha, beta-unsaturated compound.

  9. 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.

  10. 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…

  11. A structurally-characterized NbCl5-NHC adduct.

    PubMed

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

    2014-05-01

    The selective reactions of niobium pentachloride with two bulky NHC carbenes afforded NbCl5(NHC) complexes, bearing the highest oxidation state ever found for a metal centre in a transition metal halide-NHC adduct. The X-ray structure of 2a is the first one reported for a monodentate NHC-niobium species, and exhibits an abnormally long Nb-C bond. PMID:24658260

  12. Toxicological significance of DNA adducts: summary of discussions with an expert panel.

    PubMed

    Nestmann, E R; Bryant, D W; Carr, C J

    1996-08-01

    A workshop was held to discuss the uses of data on DNA adduct measurement in humans and in experimental systems in vitro and in vivo. The discussions focused principally on the understanding of the toxicological significance of DNA adducts as provided by information from animal models. An Expert Panel concluded that human DNA adduct data have utility in several aspects of risk assessment. The presence and amount of specific adducts that can be correlated with a chemical exposure are relevant for hazard identification and risk evaluation. Data from experimental systems have established dose-response relationships between the level of adducts and exposure, but these remain complex and depend on metabolic fate. Although structure-activity relationships have been useful retrospectively to explain the DNA-reactive nature of some chemicals or classes of chemicals, there are currently no means outside the laboratory to specifically predict the adduct-producing potency of a compound. Analysis of DNA adducts in tissues of laboratory animals and humans has revealed sensitive subpopulations, a finding that has important relevance for human risk assessment. Adduct analysis may be one of the best tools available to characterize exposures to DNA from complex mixtures for purposes of epidemiological investigation. Consensus statements were developed based on presentations by R. Gupta, W. Lutz, R. Nath, and B. Singer [see Regul. Toxicol. Pharmacol. 23(1), 1996] and subsequent discussions. First, rigorous scientific criteria should be met for the detection and characterization of specific DNA adducts in vitro and in target tissues in vivo. Second, the use of adduct data in risk extrapolation has the greatest value when there is characterization of adduct structure, an understanding of the role of repair in DNA adduct removal, and demonstration of biological relevance for each adduct. Third, the detection of DNA adducts in a tissue does not necessarily indicate a specific

  13. Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles.

    PubMed

    Orton, Christopher R; Liebler, Daniel C

    2007-06-30

    Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here, we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately two- to three-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity.

  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. 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.

  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. Analysis of cytogenetic effects and DNA adduct formation induced by safrole in Chinese hamster lung cells.

    PubMed

    Daimon, H; Sawada, S; Asakura, S; Sagami, F

    1997-01-01

    Safrole (1-allyl-3,4-methylenedioxybenzene) was tested for its ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) and to form DNA adducts in Chinese hamster lung (CHL) cells, in order to investigate the relationship between cytogenetic effects and DNA adduct formation under the same treatment conditions. The cells were treated with 0.025-0.2 mg/ml safrole in the presence or absence of rat liver postmitochondrial supernatant fraction (S9). Safrole induced significant SCEs and CAs dose-dependently in the presence of S9. SCEs ranged in number from 15.6 to 21.1 SCEs/cell and CAs were observed in 4-37% of cells. Using the 32P-postlabeling assay, two major and two minor safrole-DNA adducts were detected in DNA digests obtained from CHL cells in the presence of S9. The levels of total DNA adducts ranged from 1.3 to 22.8 adducts/10(7) nucleotides. The two major adducts were shown to be guanine derivatives since these adducts comigrated on polyethylenimine plates with the adducts produced by the reaction of safrole with 2'-deoxyguanosine 3'-monophosphate. A correlation was seen between DNA adducts and SCEs or CAs. Neither induction of SCEs and CAs nor formation of DNA adducts was observed in the absence of S9. These findings suggest that SCEs and CAs induced by safrole result from covalent DNA modification metabolically activated by S9 in cultured cells.

  18. 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

  19. Bulky DNA adducts, 4-aminobiphenyl-haemoglobin adducts and diet in the European Prospective Investigation into Cancer and Nutrition (EPIC) prospective study.

    PubMed

    Peluso, Marco; Airoldi, Luisa; Munnia, Armelle; Colombi, Alessandro; Veglia, Fabrizio; Autrup, Herman; Dunning, Alison; Garte, Seymour; Gormally, Emmanuelle; Malaveille, Christian; Matullo, Giuseppe; Overvad, Kim; Raaschou-Nielsen, Ole; Clavel-Chapelon, Francoise; Linseisen, Jacob; Boeing, Heiner; Trichopoulou, Antonia; Palli, Domenico; Krogh, Vittorio; Tumino, Rosario; Panico, Salvatore; Bueno-De-Mesquita, Bas H; Peeters, Petra H; Kumle, Merethe; Agudo, Antonio; Martinez, Carmen; Dorronsoro, Miren; Barricarte, Aurelio; Tormo, Marìa Jose; Quiros, José Ramón; Berglund, Goran; Jarvholm, Bengt; Day, Nicholas E; Key, Timothy J; Saracci, Rodolfo; Kaaks, Rudolf; Riboli, Elio; Bingham, Shelia; Vineis, Paolo

    2008-09-01

    In contrast to some extensively examined food mutagens, for example, aflatoxins, N-nitrosamines and heterocyclic amines, some other food contaminants, in particular polycyclic aromatic hydrocarbons (PAH) and other aromatic compounds, have received less attention. Therefore, exploring the relationships between dietary habits and the levels of biomarkers related to exposure to aromatic compounds is highly relevant. We have investigated in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort the association between dietary items (food groups and nutrients) and aromatic DNA adducts and 4-aminobiphenyl-Hb adducts. Both types of adducts are biomarkers of carcinogen exposure and possibly of cancer risk, and were measured, respectively, in leucocytes and erythrocytes of 1086 (DNA adducts) and 190 (Hb adducts) non-smokers. An inverse, statistically significant, association has been found between DNA adduct levels and dietary fibre intake (P = 0.02), vitamin E (P = 0.04) and alcohol (P = 0.03) but not with other nutrients or food groups. Also, an inverse association between fibre and fruit intake, and BMI and 4-aminobiphenyl-Hb adducts (P = 0.03, 0.04, and 0.03 respectively) was observed. After multivariate regression analysis these inverse correlations remained statistically significant, except for the correlation adducts v. fruit intake. The present study suggests that fibre intake in the usual range can modify the level of DNA or Hb aromatic adducts, but such role seems to be quantitatively modest. Fibres could reduce the formation of DNA adducts in different manners, by diluting potential food mutagens and carcinogens in the gastrointestinal tract, by speeding their transit through the colon and by binding carcinogenic substances.

  20. Zinc acetylacetonate hydrate adducted with nitrogen donor ligands: Synthesis, spectroscopic characterization, and thermal analysis

    NASA Astrophysics Data System (ADS)

    Brahma, Sanjaya; Shivashankar, S. A.

    2015-12-01

    We report synthesis, spectroscopic characterization, and thermal analysis of zinc acetylacetonate complex adducted by nitrogen donor ligands, such as pyridine, bipyridine, and phenanthroline. The pyridine adducted complex crystallizes to monoclinic crystal structure, whereas other two adducted complexes have orthorhombic structure. Addition of nitrogen donor ligands enhances the thermal property of these complexes as that with parent metal-organic complex. Zinc acetylacetonate adducted with pyridine shows much higher volatility (106 °C), decomposition temperature (202 °C) as that with zinc acetylacetonate (136 °C, 220 °C), and other adducted complexes. All the adducted complexes are thermally stable, highly volatile and are considered to be suitable precursors for metal organic chemical vapor deposition. The formation of these complexes is confirmed by powder X-ray diffraction, Fourier transform infrared spectroscopy, mass spectroscopy, and elemental analysis. The complexes are widely used as starting precursor materials for the synthesis of ZnO nanostructures by microwave irradiation assisted coating process.

  1. Charge transfer adducts of metal complexes of π-donor ligands with I 2 and TCNQ

    NASA Astrophysics Data System (ADS)

    Bera, T. R.; Sen, D.; Ghosh, R.

    1989-01-01

    Copper(II) and nickel(II) biguanides and O-alkyl-1-amidinourea can act as donors for the formation of charge transfer (CT) adducts with I 2 and tetracyanoquinodimethane (TNCQ) as acceptors. Iodine adducts are characterized both in solid and solution states whereas TCNQ adducts obtain only in solution. Appearance of a broad band at 355 nm for iodine adducts and at 335 nm for TNCQ adducts and shifting of i.r. frequencies support the formation of donor acceptor associates. Elemental analysis establishes 1:1 stoichiometry of the solid adducts. The K and ɛ values determined by modified Benesi—Hildebrand, Scott and Rose—Drago equations are found to be of the order of 10 4 and 10 3 respectively at 298 K in methanol. The solvent effect on the K values is discussed in terms of coupled solute-solute and solute-solvent equilibria.

  2. 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.

  3. 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

  4. Chromatographic and fluorescence spectroscopic studies of individual 7,12-dimethylbenz(a)anthracene--deoxyribonucleoside adducts

    SciTech Connect

    Moschel, R.C.; Pigott, M.A.; Costantino, N.; Dipple, A.

    1983-09-01

    Compared with standard Sephadex LH-20 column chromatography, a newly developed high pressure liquid chromatographic separation of hydrocarbon deoxyribonucleoside adducts derived from the DNA of mouse embryo cell cultures exposed to 7,12-dimethylbenz(a)anthracene (DMBA) provides markedly superior resolution. Once resolved, the fluorescence spectroscopic properties of the three major DMBA--DNA adducts indicate that the fluorescence exhibited by adducts derived from a bay region syn dihydrodiol epoxide of DMBA differs subtly from that exhibited by adducts derived from the isomeric anti dihydrodiol epoxide.

  5. 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.

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

    PubMed

    Lee, Soon-Hyuck; Lee, Jin-Hyuck; 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.

  7. Comparison of DNA adducts from exposure to complex mixtures in various human tissues and experimental systems

    PubMed Central

    Lewtas, Joellen; Mumford, Judy; Everson, Richard B.; Hulka, Barbara; Wilcosky, Tim; Kozumbo, Walter; Thompson, Claudia; George, Michael; Dobiáš, Lubomir; Šrám, Radim; Li, Xueming; Gallagher, Jane

    1993-01-01

    DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 4. PMID:8319665

  8. Dynamic Adduction Angle of Forefoot Measured With a Novel Technique And Its Relationship With Functional Outcomes

    PubMed Central

    Amin, Nirav Hasmukh; Jakoi, Andre; Alexander, Volpi MS; Morrison, Martin Joseph; Trobisch, Per

    2016-01-01

    Background Idiopathic clubfoot is commonly treated with the Ponseti method with the extent of invasive treatment involving tendon-Achilles lengthening. Forefoot adduction is a common complication in surgically treated clubfeet. Yet, no method has been described to measure dynamic (walking) forefoot adduction. The aim of this study was to assess the persistent pes adductus in children whose clubfeet were surgically treated using a dorsomedial soft tissue release and to find out correlations between forefoot adduction and clinical outcome measures. Methods We analysed the dynamic adduction angle in 33 clubfeet using a pressure-sensitive foot platform and compared it to the healthy feet of an age- and weight-matched group of children without congenital foot deformities. The clinical outcome was analysed using the McKay score. Results Mean dynamic adduction angle was 4.1o in the surgically corrected clubfeet, whereas it was 6.4° in unaffected feet of patients with unilateral clubfoot and 7.1o in control group. The McKay score were excellent in 1 patient, good in 5, average in 13, and fair in 4 of the 23 patients. There was no correlation between dynamic adduction angle and McKay score using paired t test (P > 0.05). Conclusion High occurrence of dynamic adduction angle in surgically treated clubfeet was detected. In conclusion, no correlation between forefoot adduction, dynamic forefoot adduction angle and clinical outcome measures within the study was observed. PMID:27547113

  9. 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

  10. TRPA1-mediated accumulation of aminoglycosides in mouse cochlear outer hair cells.

    PubMed

    Stepanyan, Ruben S; Indzhykulian, Artur A; Vélez-Ortega, A Catalina; Boger, Erich T; Steyger, Peter S; Friedman, Thomas B; Frolenkov, Gregory I

    2011-12-01

    Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for

  11. 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. PMID:19253935

  12. Escherichia coli responses to a single DNA adduct.

    PubMed

    Pandya, G A; Yang, I Y; Grollman, A P; Moriya, M

    2000-12-01

    To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed. PMID:11073901

  13. Detection of mitomycin C-DNA adducts in vivo by 32P-postlabeling: time course for formation and removal of adducts and biochemical modulation.

    PubMed

    Warren, A J; Maccubbin, A E; Hamilton, J W

    1998-02-01

    Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed 32P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N2G-MMC-N2G interstrand cross-link, the bifunctionally activated MMC-N2G monoadduct, and two isomers (alpha and beta) of the monofunctionally activated MMC-N2G monoadduct. The (GpG) N2G-MMC-N2G intrastrand cross-link appears to be a poor substrate for nuclease P1 and/or T4 kinase and was not evaluable by this assay. Levels of all four detectable adducts increased substantially within the first 2 h after MMC treatment, reached maximal levels by 6 h, and decreased progressively thereafter through 24 h, although low levels of certain adducts persisted beyond 24 h. Lung and kidney had comparable levels of total MMC adducts, which were approximately 60% those of the liver, and there were no significant differences in the proportion of specific adducts among the three tissues. The interstrand cross-link represented approximately 13-14% of the total MMC adducts, which is approximately 5-fold greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relative to the three monoadducts, suggesting preferential repair. The effect of modulating different components of the Phase I and Phase II drug metabolism on MMC adduct formation, using either glutethimide, 3,4,3',4'-tetrachlorobiphenyl, dexamethasone, buthionine sulfoximine, ethacrynic acid, or N-acetylcysteine pretreatments, was

  14. 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

  15. Age-dependent increase of etheno-DNA-adducts in liver and brain of ROS overproducing OXYS rats

    SciTech Connect

    Nair, Jagadeesan; Sinitsina, Olga; Vasunina, Elena A.; Nevinsky, Georgy A.; Laval, Jacques; Bartsch, Helmut . E-mail: h.bartsch@dkfz.de

    2005-10-21

    Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N {sup 6}-ethenodeoxyadenosine ({epsilon}dA) and 3,N {sup 4}-ethenodeoxycytidine ({epsilon}dC) by immunoaffinity/{sup 32}P-postlabelling. While {epsilon}dA and {epsilon}dC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for {epsilon}dA by immunostaining of nuclei. Brains from old OXYS rats accumulated {epsilon}dA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.

  16. DNA adduct-induced stabilization of slipped frameshift intermediates within repetitive sequences: implications for mutagenesis.

    PubMed Central

    Garcia, A; Lambert, I B; Fuchs, R P

    1993-01-01

    Chemical carcinogens such as the aromatic amide 2-acetylaminofluorene (AAF) are known to induce -1 frameshift mutation hotspots at repetitive sequences. This mutagenesis pathway was suggested to involve slipped intermediates formed during replication. To investigate the stability and structure of such intermediates we have constructed DNA duplexes containing single AAF adducts within a run of three guanine residues. The strand complementary to that bearing the AAF adducts contained either the wild-type sequence (homoduplexes) or lacked one cytosine directly opposite the run of guanines containing the AAF adduct and thus modeled the putative slipped mutagenic intermediates (SMIs). The melting temperature of AAF-modified homoduplexes or the unmodified SMI was reduced by approximately 10 degrees C relative to the unmodified homoduplex. Surprisingly, AAF adducts stabilized the SMIs as evidenced by an increase in melting temperature to a level approaching that of the unmodified homoduplex. The chemical probes hydroxylamine and bromoacetaldehyde were strongly reactive toward cytosine residues opposite the adduct in AAF-modified homoduplexes, indicating adduct-induced denaturation. In contrast, no cytosine reactivities were observed in the AAF-modified SMIs, suggesting that the two cytosines were paired with unmodified guanines. Use of diethyl pyrocarbonate to probe the guanine residues showed that all three guanines in the unmodified SMI adopted a transient single-stranded state which was delocalized along the repetitive sequence. However, when an AAF adduct was present, reduced diethyl pyrocarbonate reactivity at guanines adjacent to the adduct in AAF-modified SMIs reflected localization of the bulge to the adducted base. Our results suggest that AAF exerts a local denaturing and destabilizing effect within the homoduplex which is alleviated by the formation of a bulge. The stabilization by the AAF adduct of the SMIs may contribute to the dramatic increase in -1

  17. Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis

    PubMed Central

    Gounarides, John; Cobb, Jennifer S.; Zhou, Jing; Cook, Frank; Yang, Xuemei; Yin, Hong; Meredith, Erik; Rao, Chang; Huang, Qian; Xu, YongYao; Anderson, Karen; De Erkenez, Andrea; Liao, Sha-Mei; Crowley, Maura; Buchanan, Natasha; Poor, Stephen; Qiu, Yubin; Fassbender, Elizabeth; Shen, Siyuan; Woolfenden, Amber; Jensen, Amy; Cepeda, Rosemarie; Etemad-Gilbertson, Bijan; Giza, Shelby; Mogi, Muneto; Jaffee, Bruce; Azarian, Sassan

    2014-01-01

    Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others. PMID:25343517

  18. 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

  19. Specific plant DNA adducts as molecular biomarkers of genotoxic atmospheric environments.

    PubMed

    Weber-Lotfi, F; Obrecht-Pflumio, S; Guillemaut, P; Kleinpeter, J; Dietrich, A

    2005-03-01

    The general purpose of this study was to determine whether the formation of DNA addition products ('adducts') in plants could be a valuable biomarker of genotoxic air pollution. Plants from several species were exposed to ambient atmosphere at urban and suburban sites representative of different environmental conditions. The levels of NO2 and of the quantitatively major genotoxic air pollutants benzene, toluene, and xylene were monitored in parallel with plant exposure. DNA adducts were measured in bean (Phaseolus vulgaris), rye-grass (Lolium perenne), and tobacco (Nicotiana tabacum) seedlings by means of the [32P]-postlabeling method. Whereas, no correlation was found between the levels of the major genotoxic air pollutants and the total amounts of DNA adducts, individual analyses revealed site-specific and plant species-specific adduct responses, both at the qualitative and quantitative level. Among these, the amount of a specific rye-grass DNA adduct (rgs1) correlated with benzene/toluene/xylene levels above a threshold. For further characterization, rye-grass seedlings were treated in controlled conditions with benzene, toluene, xylene or their derivatives. On the other hand, in vitro DNA adduct formation assays were developed involving benzene, toluene, xylene, or their derivatives, and plant microsomes or purified peroxidase. Although in some cases, these approaches produced specific adduct responses, they failed to generate the rgs1 DNA adduct, which appeared to be characteristic for on-site test-plant exposure. Our studies have thus identified an interesting candidate for further analysis of environmental biomarkers of genotoxicity.

  20. 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...

  1. 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...

  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. gamma. Irradiation induced formation of PCB-solvent adducts in aliphatic solvents

    SciTech Connect

    Lepine, F.; Milot, S.; Gagne, N. )

    1990-09-01

    {gamma}Irradiation induced formation of PCB-solvent adducts was investigated as a model for PCB residues in irradiated food. Formation of cyclohexyl adducts of PCBs was found to be significant when pure PCB congeners and Aroclor mixture were irradiated in cyclohexane and cyclohexene. Reaction pathways were investigated, and the effects of oxygen and electron scavenger were studied.

  4. A hydrogen bond scaffold supported synthetic heme Fe(III)-O2(-) adduct.

    PubMed

    Mittra, Kaustuv; Chatterjee, Sudipta; Samanta, Subhra; Sengupta, Kushal; Bhattacharjee, Hridaynath; Dey, Abhishek

    2012-11-01

    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.

  5. Cyclooctyne [60]fullerene hexakis adducts: a globular scaffold for copper-free click chemistry.

    PubMed

    Ramos-Soriano, Javier; Reina, José J; Pérez-Sánchez, Alfonso; Illescas, Beatriz M; Rojo, Javier; Martín, Nazario

    2016-08-18

    The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields. This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications. PMID:27492263

  6. 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

  7. Identification of polycyclic aromatic hydrocarbon metabololites and DNA adducts in mixtures using fluorescence line narrowing spectrometry

    SciTech Connect

    Sanders, M.J.; Cooper, R.S.; Jankowiak, R.; Small, G.J.; Heisig, V.; Jeffrey, A.M.

    1986-04-01

    Fluorescence line narrowing spectrometry is applied to five modifications of DNA (intact adducts) formed from diol epoxides of benzo(a)pyrene, chrysene, 5-methylchrysene, and benz(a)anthracene. The direct identification of all five adducts in a laboratory mixture is accomplished. In addition, a mixture of six corresponding metabolites plus the DNA adducts from benzo(a)pyrene and 5-methylchrysene is resolved. Each adduct can be distinguished from its corresponding tetrol metabolite. Utilization of an intensified diode array-optical multichannel analyzer provides detection of the adduct from benzo(a)pyrene with a S/N approx. 150 for a damage level of approx. 5 bases in 10/sup 6/. 25 references, 7 figures.

  8. 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.

  9. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts

    PubMed Central

    Aparicio, Tomas; Baer, Richard; Gottesman, Max

    2016-01-01

    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. PMID:26880199

  10. 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 7{beta}, 8{alpha}-dihydoxy-9{alpha}, l0{alpha}-epoxy-7,8,9, 10-tetrahydrobenzo[{alpha}]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, ({minus})-trans-, (+)-cis- and ({minus})-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 ( {approximately} 25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant {pi}-{pi} 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 G{sub 2} or G{sub 3} (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-N{sup 2}-dG in DNA isolated from the skin of mice treated topically with benzo[{alpha}]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-N{sup 2}-dG.

  11. 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.

  12. /sup 32/P-postlabeling analysis of aromatic DNA adducts in fish from polluted areas

    SciTech Connect

    Dunn, B.P.; Black, J.J.; Maccubbin, A.

    1987-12-15

    Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using /sup 32/P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to /sup 32/P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and /sup 32/P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.

  13. Depurinating estrogen-DNA adducts, generators of cancer initiation: their minimization leads to cancer prevention.

    PubMed

    Cavalieri, Ercole L; Rogan, Eleanor G

    2016-03-01

    Estrogens can initiate cancer by reacting with DNA. Specific metabolites of endogenous estrogens, the catechol estrogen-3,4-quinones, react with DNA to form depurinating estrogen-DNA adducts. Loss of these adducts leaves apurinic sites in the DNA, generating mutations that can lead to the initiation of cancer. A variety of endogenous and exogenous factors can disrupt estrogen homeostasis, which is the normal balance between estrogen activating and protective enzymes. In fact, if estrogen metabolism becomes unbalanced and generates excessive catechol estrogen 3,4-quinones, formation of depurinating estrogen-DNA adducts increases and the risk of initiating cancer is greater. The levels of depurinating estrogen-DNA adducts are high in women diagnosed with breast cancer and those at high risk for the disease. High levels of depurinating estrogen-DNA adducts before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Women with thyroid or ovarian cancer also have high levels of estrogen-DNA adducts, as do men with prostate cancer or non-Hodgkin lymphoma. Depurinating estrogen-DNA adducts are initiators of many prevalent types of human cancer. These findings and other discoveries led to the recognition that reducing the levels of estrogen-DNA adducts could prevent the initiation of human cancer. The dietary supplements N-acetylcysteine and resveratrol inhibit formation of estrogen-DNA adducts in cultured human breast cells and in women. These results suggest that the two supplements offer an approach to reducing the risk of developing various prevalent types of human cancer. Graphical abstract Major metabolic pathway in cancer initiation by estrogens. PMID:26979321

  14. 32P-postlabeling analysis of aromatic DNA adducts in fish from polluted areas.

    PubMed

    Dunn, B P; Black, J J; Maccubbin, A

    1987-12-15

    Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using 32P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to 32P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and 32P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.

  15. 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.

  16. Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

    PubMed

    Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

    2015-12-15

    4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.

  17. 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.

  18. 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...

  19. Absence of formation of benzo[a]pyrene/DNA adducts in the cuttlefish (Sepia officinalis, Mollusca: Cephalopoda)

    SciTech Connect

    Lee, P.G.; Lu, L.J.W.; Salazar, J.J.; Holoubek, V. )

    1994-01-01

    Benzo[a]pyrene (B[a]P) injected intramuscularly into the base of the arms of cuttlefish was released continuously from the injection site and removed from the organism. Only a portion of the compound accumulated in the body. Twenty-four hr after its injection, 75% of B[a]P applied in olive oil was removed from the cuttlefish, and 1.2% was found in the body outside the head, in site of injection. If the carcinogen was dissolved in dimethylformamide, the removal of B[a]P was slower, so that only 18% of the injected B[a]P was removed from the organism and 0.36% accumulated in the body outside the head 24 hr after injection. The high level of B[a]P in gills and hemolymph 4 hr after injection and the kinetics of the decrease of its concentration with time indicate that these two organs could be involved in the excretion of B[a]P from the body. The B[a]P/DNA adducts characteristic for vertebrates could not be demonstrated in gills, skin, brain, hepatopancreas, and lymphocytes of the cuttlefish 24 hr after injection. The dose of the carcinogene injected into the cuttlefish was 2-4 times higher than the dose resulting in the formation of a high level of B[a]P/DNA adducts in vertebrates. A different metabolism of B[a]P in the tissue of cephalopods, compared to vertebrates, could be less favorable to the process leading to malignant transformation and could explain the absence from the literature of reports of tumors in cephalopods. 15 refs., 1 fig., 2 tabs.

  20. 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.

  1. 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.

  2. Detection and characterization of human serum antibodies to polycyclic aromatic hydrocarbon diol-epoxide DNA adducts

    SciTech Connect

    Newman, M.J.; Light, B.A.; Weston, A.; Tollurud, D.; Clark, J.L.; Mann, D.L.; Blackmon, J.P.; Harris, C.C.

    1988-07-01

    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.

  3. 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

  4. Hemoglobin adducts of N-substituted aryl compounds in exposure control and risk assessment.

    PubMed

    Neumann, H G; Birner, G; Kowallik, P; Schütze, D; Zwirner-Baier, I

    1993-03-01

    Arylamines, nitroarenes, and azo dyes yield a common type of metabolite, the nitroarene, which produces a hydrolyzable adduct with protein and is closely related to the critical, ultimate toxic and genotoxic metabolite. The target dose as measured by hemoglobin adducts in erythrocytes reflects not only the actual uptake from the environment but also an individual's capacity for metabolic activation and is therefore an improved dosimeter for human exposure. The usefulness of hemoglobin adducts in molecular epidemiology is now widely recognized. With regard to risk assessment, many questions need to be answered. The described experiments in rats address some of these questions. The relationship between binding to hemoglobin in erythrocytes and to proteins in plasma has been found to vary considerably for a number of diamines. The fraction of hydrolyzable adducts out of the total protein adducts formed also varies in both compartments. This indicates that the kind of circulating metabolites and their availability in different compartments is compound specific. This has to do with the complex pattern of competing metabolic pathways, and the role of N-acetylation and deacetylation is emphasized. An example of nonlinear dose dependence adds to the complexity. Analysis of hemoglobin adducts reveals interesting insights into prevailing pathways, which not only apply to the chemical, but may also be useful to assess an individual's metabolic properties. In addition, it is demonstrated that the greater part of erythrocytes and benzidine-hemoglobin adducts are eliminated randomly in rats, i.e., following first-order kinetics.

  5. 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.

  6. Bulky DNA adducts in human sperm: relationship with fertility, semen quality, smoking, and environmental factors.

    PubMed

    Horak, Stanislaw; Polanska, Joanna; Widlak, Piotr

    2003-05-01

    The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.

  7. 32P-postlabeling detection of DNA adducts in fish from chemically contaminated waterways.

    PubMed

    Maccubbin, A E; Black, J J; Dunn, B P

    1990-05-01

    Fish were collected from sites in the chemically-contaminated Buffalo River, New York, and the Detroit River, Michigan. The sediments of these rivers have high levels of chemical contaminants, including polycyclic aromatic hydrocarbons (PAHs), and fish from these locations have high prevalences of liver cancer. To determine chemical-DNA interactions and a possible role for chemicals as a cause of the observed tumors, DNA was isolated from livers and was enzymatically digested to normal and adducted nucleotides. The DNA digests were enriched for hydrophobic, bulky adducts, either by preparative reverse phase high pressure liquid chromatography, or by selective nuclease P1 dephosphorylation of normal nucleotides. DNA-chemical adducts were then quantitated by 32P-postlabeling analysis. Regardless of the adduct enrichment procedure, the chromatograms derived from DNA of fish from polluted areas showed a diffuse, diagonal radioactive zone consisting, at least in part, of multiple overlapping discrete adduct spots. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky, hydrophobic, aromatic genotoxic compounds. Analysis of bile demonstrated recent exposure to multi-ringed aromatic compounds.

  8. 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.

  9. 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

  10. 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.

  11. DNA adducts in human pancreatic tissues and their potential role in carcinogenesis.

    PubMed

    Wang, M; Abbruzzese, J L; Friess, H; Hittelman, W N; Evans, D B; Abbruzzese, M C; Chiao, P; Li, D

    1998-01-01

    Pancreas cancer is the fourth and fifth leading cause of cancer death for men and women, respectively, in the United States. Although the etiology of this cancer is poorly understood, smoking and dietary fat have been implicated by epidemiological studies. To test the hypothesis that DNA damage derived from carcinogen exposure and diet is involved in pancreatic carcinogenesis, aromatic and lipid peroxidation-related DNA adducts in 13 normal tissues adjacent to tumor and 20 tumors from pancreatic cancer patients were analyzed by 32P-postlabeling. Normal pancreatic tissues from 5 nonpancreatic cancer patients and 19 healthy organ donors served as controls. To correlate the DNA adduct level with patients' characteristics, information on age, sex, body mass index, and smoking status of pancreatic cancer patients were collected from medical records. A significantly higher level of total DNA adducts was detected in pancreatic cancer patients as compared with controls. The mean level of adducts/10(8) nucleotides in adjacent normal pancreatic tissues from pancreatic cancer patients (A tissues) was 102 +/- 21 compared with 39 +/- 6 and 13 +/- 1 in pancreatic tumor tissues (T tissues) and normal pancreatic tissues from controls (C tissues), respectively. Among the adducts observed, one single aromatic adduct (spot 1) was present in 100, 90, and 0% of the A, T, and C tissues, respectively. Two novel clusters of adducts (spots 2 and 3) were observed in 11 of 13, 12 of 20, and 2 of 24 of A, T, and C tissues, respectively, and the presence of these adducts was positively correlated with smoking status. In addition, the previously defined smoking-related diagonal radioactive zone was detected in three A samples only, although 50% (10 of 20) of the patients with pancreatic cancers in this study were ever smokers. Putative lipid peroxidation-related adducts were detected in all samples examined and were significantly higher in A than in T and C samples. Multiple regression analyses

  12. 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

  13. 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

  14. Adenine-DNA adducts derived from the highly tumorigenic dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not

    PubMed Central

    Kropachev, Konstantin; Kolbanovskiy, Marina; Liu, Zhi; Cai, Yuqin; Zhang, Lu; Schwaid, Adam G.; Kolbanovskiy, Alexander; Ding, Shuang; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2013-01-01

    The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N2-dG) and adenine (N6-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N2-dG linkage site is ~ 35 times more susceptible to NER dual incisions than the stereochemically identical N6-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar, but somewhat smaller effect (factor of ~15) is observed. The striking resistance of the bulky N6-dA in contrast to the modest to good susceptibilities of the N2-dG adducts to NER are interpreted in terms of the balance between lesion-induced DNA-distorting and DNA-stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA. PMID:23570232

  15. Urinary biomarkers suggest that estrogen-DNA adducts may play a role in the aetiology of non-Hodgkin lymphoma

    PubMed Central

    Gaikwad, Nilesh W.; Yang, Li; Weisenburger, Dennis D.; Vose, Julie; Beseler, Cheryl; Rogan, Eleanor G.; Cavalieri, Ercole L.

    2015-01-01

    A variety of evidence suggests that estrogens may induce non-Hodgkin lymphoma (NHL). The reaction of catechol estrogen quinones with DNA to form depurinating estrogen-DNA adducts is hypothesized to initiate this process. These adducts are released from DNA, shed from cells into the bloodstream and excreted in urine. The aim of this study was to determine whether or not the depurinating estrogen-DNA adducts might be involved in the aetiology of human NHL. Estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified in spot urine samples from 15 men with NHL and 30 healthy control men by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of estrogen-DNA adducts were significantly higher in the men with NHL than in the healthy control men. Thus, formation of estrogen-DNA adducts may play a critical role in the aetiology of NHL, and these adducts could be potential biomarkers of NHL risk. PMID:19863189

  16. Second vowel formant relationship to adduction: A preliminary study

    NASA Astrophysics Data System (ADS)

    Hanrahan, Kevin G.

    The relationship between the vocal tract and the larynx in the formation of vowels has been debated for decades. Vowels were first thought to have been formed in the larynx; then later it was believed that they were formed solely in the vocal tract. In the 1960s Fant formalized this belief into the Source-Filter Theory of Vowel Formation. The theory was interpreted by voice teachers to mean that the larynx had very little to do with the formation of vowels, and this interpretation has dominated voice teaching for decades. Recent research, however, is now suggesting that the larynx and the vocal tract are interactive with each other, meaning that a change of muscular function in the larynx will create a change of resonator function in the vocal tract, and vice versa. This conclusion is drawn mainly on the work of Titze, Story, Laukkanen, et.al. They have found that a relationship exists between laryngeal function and the first vowel formant (F1). When examining research on the second vowel formant (F2), this author discovered that there may be a relationship between F2 and adduction. Therefore, based on present evidence, it was hypothesized that an elevated frequency of F2 corresponded to an increase in adduction. The hypothesis was examined by comparing the resonance output and glottal closure between vowels where F2 was elevated and vowels without modification of F2. Subjects were asked to sing [i], [a], and [u] at a medium dynamic level on D4, G#4, and D5 for the female subjects and an octave below for the male subjects, once using a "generic" version of the vowel, meaning what they considered a "nice, easy, and generic" version of the vowel to be, and then again modifying the vowel to increase the frequency of the upper harmonics. Electroglottogram, pitch, intensity, and formant data were collected and compared. An increase in the frequency of F2 corresponded to an increase in the Closed Quotient (CQ), the length of time the vocal folds are closed, in a few

  17. Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin.

    PubMed

    Meng, Xiaoli; Maggs, James L; Usui, Toru; Whitaker, Paul; French, Neil S; Naisbitt, Dean J; Park, B Kevin

    2015-01-20

    Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.

  18. 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.

  19. A rescue act: Translesion DNA synthesis past N(2) -deoxyguanosine adducts.

    PubMed

    Nair, Deepak T; Kottur, Jithesh; Sharma, Rahul

    2015-07-01

    Genomic DNA is continually subjected to a number of chemical insults that result in the formation of modified nucleotides--termed as DNA lesions. The N(2) -atom of deoxyguanosine is particularly reactive and a number of chemicals react at this site to form different kinds of DNA adducts. The N(2) -deoxyguanosine adducts perturb different genomic processes and are particularly deleterious for DNA replication as they have a strong tendency to inhibit replicative DNA polymerases. Many organisms possess specialized dPols--generally classified in the Y-family--that serves to rescue replication stalled at N(2) -dG and other adducts. A review of minor groove N(2) -adducts and the known strategies utilized by Y-family dPols to replicate past these lesions will be presented here.

  20. Glottal configuration, acoustic, and aerodynamic changes induced by variation in suture direction in arytenoid adduction procedures.

    PubMed

    Inagi, Katsuhide; Connor, Nadine P; Suzuki, Tatsutoshi; Ford, Charles N; Bless, Diane M; Nakajima, Masami

    2002-10-01

    Arytenoid adduction is a phonosurgical procedure in which the arytenoid cartilages are approximated to reduce posterior glottal gap size and improve voice. Voice outcomes following arytenoid adduction are not always optimal. The goal of this study was to systematically vary suture direction and force of pull on the arytenoid cartilages in a human excised laryngeal model to determine the optimal combination of factors for reducing glottal gap and improving voice. Several factors demonstrated significant effects. Changes in suture direction and force of pull affected glottal configuration in both the horizontal and vertical planes. Increased force of pull on the muscular process resulted in increased adduction of the vocal process for all suture directions. Changes in suture direction and force of pull also affected acoustic and aerodynamic measures of induced voice. Therefore, voice outcomes can be optimized with arytenoid adduction if the vocal fold plane is accurately adjusted.

  1. 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.

  2. 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

  3. 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.

  4. Effect of Laterally Wedged Insoles on the External Knee Adduction Moment across Different Reference Frames

    PubMed Central

    Yamaguchi, Satoshi; Kitamura, Masako; Ushikubo, Tomohiro; Murata, Atsushi; Akagi, Ryuichiro; Sasho, Takahisa

    2015-01-01

    Objective Biomechanical effects of laterally wedged insoles are assessed by reduction in the knee adduction moment. However, the degree of reduction may vary depending on the reference frame with which it is calculated. The purpose of this study was to clarify the effect of reference frame on the reduction in the knee adduction moment by laterally wedged insoles. Methods Twenty-nine healthy participants performed gait trials with a laterally wedged insole and with a flat insole as a control. The knee adduction moment, including the first and second peaks and the angular impulse, were calculated using four different reference frames: the femoral frame, tibial frame, laboratory frame and the Joint Coordinate System. Results There were significant effects of reference frame on the knee adduction moment first and second peaks (P < 0.001 for both variables), while the effect was not significant for the angular impulse (P = 0.84). No significant interaction between the gait condition and reference frame was found in either of the knee adduction moment variables (P = 0.99 for all variables), indicating that the effects of laterally wedged insole on the knee adduction moments were similar across the four reference frames. On the other hand, the average percent changes ranged from 9% to 16% for the first peak, from 16% to 18% for the second peak and from 17% to 21% for the angular impulse when using the different reference frames. Conclusion The effects of laterally wedged insole on the reduction in the knee adduction moment were similar across the reference frames. On the other hand, Researchers need to recognize that when the percent change was used as the parameter of the efficacy of laterally wedged insole, the choice of reference frame may influence the interpretation of how laterally wedged insoles affect the knee adduction moment. PMID:26397375

  5. Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay

    PubMed Central

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-01-01

    Enzymes that form transient DNA–protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA–protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA–protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA–protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA–protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA–protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1–DNA and Top2a–DNA adducts in human cells, and gyrase–DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  6. Determinants of the variability of aflatoxin-albumin adduct levels in Ghanaians.

    PubMed

    Dash, B; Afriyie-Gyawu, E; Huebner, H J; Porter, W; Wang, J S; Jolly, P E; Phillips, T D

    2007-01-01

    Hepatocellular carcinoma (HCC) is a multifactorial disease with various host and environmental factors involved in its etiology. Of these, aflatoxin exposure has been established as an important risk factor in the development of HCC; the presence of aflatoxin-albumin (AA) adducts in the blood serves as a valuable biomarker of human exposure. In this study, the relationship between a variety of different HCC host factors and the incidence of AA adduct levels was examined in a Ghanaian population at high risk for HCC. These factors included age, gender, hepatitis virus B (HVB) and hepatitis C virus (HCV) status, and genetic polymorphisms in both microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). Blood samples were analyzed for AA adducts and HBV and HCV status. GSTM1 and GSTT1 deletion polymorphisms and mEH exon 3 and exon 4 single-nucleotide polymorphisms (SNPs) were determined from urine samples. In univariate analysis, age, HBV and HVC status, and GSTT1 and mEH exon 3 genotypes were not associated with AA adduct levels. However, mean adduct levels were significantly higher in both females and individuals typed heterozygous for mEH exon 4 (vs. wild types). Stratification analysis also showed that gender along with mEH exon 4 genotype and HBV status had a significant effect on adduct levels. Both females typed HBsAg+ and males with mEH exon 4 heterozygote genotypes showed significantly higher adduct levels as compared to the HBsAg- and wild types, respectively. Understanding the relationships between these host factors and the variability in aflatoxin-adduct levels may help in identifying susceptible populations in developing countries and for targeting specific public health interventions for the prevention of aflatoxicoses in populations with HCC and chronic liver diseases. PMID:17162498

  7. Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase γ.

    PubMed

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

    2013-05-17

    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,N(2)-propano-2'-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N(6)-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.

  8. 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

  9. [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.

  10. 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.

  11. 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.

  12. 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

  13. Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli

    SciTech Connect

    Burnouf, D.; Fuchs, R.P.P. ); Gauthier, C.; Chottard, J.C. )

    1990-08-01

    The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP(d(ApG)) adducts, although they account for only 25% of the lesions formed are {approx}5 times more mutagenic than the major GG adduct. The authors report the construction of vectors bearing a single cisDDP(d(ApG)) lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5{prime} base of the adduct. Single A {yields} T transversions are mainly observed (80%), whereas A {yields} G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5{prime} to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP(d(ApG)) adducts are not blocking lesions. The high mutation specificity of cisDDP-(d(ApG))-induced mutagenesis is discussed in relation to structural data.

  14. 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

  15. 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. PMID:25427902

  16. 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

  17. Scavenging of Toxic Acrolein by Resveratrol and Hesperetin and Identification of Adducts.

    PubMed

    Wang, Weixin; Qi, Yajing; Rocca, James R; Sarnoski, Paul J; Jia, Aiqun; Gu, Liwei

    2015-11-01

    The objective of this study was to investigate the ability of resveratrol and hesperetin to scavenge acrolein at pH 7.4 and 37 °C. About 6.4 or 5.2% of acrolein remained after reaction with resveratrol or hesperetin for 12 h at equimolar concentrations. An acrolein-resveratrol adduct and two acrolein-hesperetin adducts were isolated. Their structures were elucidated using mass and NMR spectroscopy. Acrolein reacted with resveratrol at the C-2 and C-3 positions through nucleophilic addition and formed an additional heterocyclic ring. Two similar monoacrolein-conjugated adducts were identified for hesperetin. Spectroscopic data suggested each acrolein-hesperetin adduct was a mixture of four stereoisomers due to the existence of two chiral carbon atoms. Yield of adducts was low at pH 5.4 but increased at pH 7.4 and 8.4. Higher pH also promoted the formation of diacrolein adducts. Results suggest that resveratrol and hesperetin exert health benefits in part through neutralizing toxic acrolein in vivo.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Potential Therapeutic Advantages of Doxorubicin when Activated by Formaldehyde to Function as a DNA Adduct-Forming Agent.

    PubMed

    Cutts, Suzanne M; Rephaeli, Ada; Nudelman, Abraham; Ugarenko, Michal; Phillips, Don R

    2015-01-01

    Doxorubicin has been in use as a key anticancer drug for forty years, either as a single agent or in combination chemotherapy. It functions primarily by interfering with topoisomerase II activity but in the presence of formaldehyde, it forms adducts with DNA, mainly with the exocyclic amine of guanine at GpC sites and these adducts are more cytotoxic than topoisomerase II induced damage. High levels of adducts form spontaneously from the endogenous level of formaldehyde in tumour cells (1,300 adducts per cell after a 4 hr treatment with doxorubicin), but substantially higher levels form with the addition of exogenous sources of formaldehyde, such as formaldehyde releasing prodrugs. The enhanced cytotoxicity of adducts has been confirmed in mouse models, with adduct-forming conditions resulting in much improved inhibition of tumour growth, as well as cardioprotection. Doxorubicin cardiotoxicity has been attributed to topoisomerase II poisoning, and the cardioprotection is consistent with a mechanism switch from topoisomerase II poisoning to covalent adduct formation. Although the adducts have a half-life of less than one day, a population remains as essentially permanent lesions. The capacity of doxorubicin to form adducts offers a range of potential advantages over the conventional use of doxorubicin (as a topoisomerase II poison), including: enhanced cell kill; tumour-selective activation, hence tumour-selective cell kill; decreased cardiotoxicity; decreased resistance to prolonged doxorubicin treatment. There is therefore enormous potential to improve clinical responses to doxorubicin by using conditions which favour the formation of doxorubicin-DNA adducts.

  4. The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts.

    PubMed

    Wyss, Laura A; Nilforoushan, Arman; Williams, David M; Marx, Andreas; Sturla, Shana J

    2016-08-19

    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 O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) 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 O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies. PMID:27378785

  5. Benzo(a)pyrene-albumin adducts in humans exposed to polycyclic aromatic hydrocarbons in an industrial area of Poland.

    PubMed Central

    Kure, E H; Andreassen, A; Ovrebø, S; Grzybowska, E; Fiala, Z; Strózyk, M; Chorazy, M; Haugen, A

    1997-01-01

    OBJECTIVES: The interaction of benzo(a)pyrene with serum albumin was measured in an attempt to identify the actual exposure and to evaluate albumin adduct measurements as biomarkers for exposure monitoring. METHODS: Benzo(a)pyrene-diol-epoxide (BPDE)-albumin adducts were measured by competitive enzyme linked immunosorbent assay (ELISA) in plasma of coke oven plant workers from three plants and from people living in a highly industrialised area of Silesia in Poland. Due to the high air concentrations of polycyclic aromatic hydrocarbons (PAHs) in this area, a control group was selected from a rural non-industrialised area in Poland. Breathing zone air measurements of PAHs were collected from some of the participants. RESULTS: Coke oven plant workers and non-occupationally exposed people had similar concentrations of albumin adducts whereas the rural controls were significantly lower (2.74 fmol adducts/microgram albumin (SEM 0.124)). The mean concentration of BPDE-albumin adduct in plasma of both the occupational and the environmental groups were significantly higher in the summer samples (4.34 fmol adducts/microgram albumin (SEM 0.335) and 4.55 fmol adducts/microgram albumin (SEM 0.296), respectively) than in the winter samples (3.06 fmol adducts/microgram albumin (SEM 0.187) and 3.04 fmol adducts/microgram albumin (SEM 0.184), respectively) even though the air measurements showed higher concentrations of PAHs in the winter. The statistical analysis did not show any effects of air exposures on concentrations of BPDE-albumin adduct. CONCLUSIONS: A multiple regression analysis of the measured concentrations of BPDE-albumin adducts for all the groups, during both seasons, indicates that occupational exposures do not contribute significantly to the formation of adducts. In general, the concentrations of albumin adducts found vary within relatively small limits for the two seasons and between the various groups of participants. No extreme differences were found. PMID

  6. 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. PMID:27486909

  7. C18 thin-layer chromatographic enhancement of the 32P-postlabeling assay for aromatic or bulky carcinogen-DNA adducts: evaluation of adduct recoveries in comparison with nuclease P1 and butanol methods.

    PubMed

    Reddy, M V

    1993-05-01

    The suitability of C18 reversed-phase thin-layer chromatography (TLC) for enrichment of adducts in the 32P-postlabeling assay was investigated for structurally diverse classes of DNA adducts derived from benzo[a]pyrene, 2-acetylaminofluorene, benzoquinone, safrole, and mitomycin C. The TLC enrichment involved retention of adducts to the C18 phase followed by elution with organic solvent-water. Adduct patterns obtained by the C18 purification were qualitatively similar to those obtained by the nuclease P1 and butanol procedures, the two commonly used enrichment methods. Adduct recoveries by the C18 method varied for different adducts and were significantly lower than those obtained by the other two techniques.

  8. New isocyanate-specific albumin adducts of 4,4'-methylenediphenyl diisocyanate (MDI) in rats.

    PubMed

    Kumar, Anoop; Dongari, Nagaraju; Sabbioni, Gabriele

    2009-12-01

    4,4'-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Albumin adducts of MDI might be involved in the etiology of sensitization reactions. It is, therefore, necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urinary metabolites or protein adducts have been used as biomarkers in workers exposed to MDI. However, with these methods it is not possible to determine whether the biomarkers result from exposure to MDI or to the parent aromatic amine 4,4'-methylenedianiline (MDA). This work presents a procedure for the determination of isocyanate-specific albumin adducts. In a long-term experiment, designed to determine the carcinogenic and toxic effects of MDI, rats were exposed chronically for 3 months, to 0.0 (control), 0.26, 0.70, and 2.06 mg MDI/m(3) as aerosols. Albumin was isolated from plasma, digested with Pronase E, and analyzed by LC-MS/MS. MDI formed adducts with lysine: N(6)-[({4-[4-aminobenzyl]phenyl}amino)carbonyl]lysine (MDI-Lys) and N(6)-[({4-[4-(acetylamino)benzyl]phenyl}amino)carbonyl] lysine (AcMDI-Lys). For the quantitation of the adducts in vivo, isotope dilution mass spectrometry was used to measure the adducts in 2 mg of albumin. The adducts found in vivo (MDI-Lys and AcMDI-Lys) and the corresponding isotope labeled compounds (MDI-[(13)C(6)(15)N(2)]Lys and Ac[(2)H(4)]MDI-Lys) were synthesized and used for quantitation. The MDI-Lys levels increased from 0-24.8 pmol/mg albumin, and the AcMDI-Lys levels increased from 0-1.85 pmol/mg albumin. The mean ratio of MDI-Lys/AcMDI-Lys for each dose level was greater than >20. The albumin adducts correlate with other biomarkers measured in the same rats in the past: urinary metabolites and hemoglobin adducts released after mild base hydrolysis. This method will enable one to

  9. Quantitation of an Acetaldehyde Adduct in Human Leukocyte DNA and the Effect of Smoking Cessation

    PubMed Central

    Chen, Li; Wang, Mingyao; Villalta, Peter W.; Luo, Xianghua; Feuer, Rachel; Jensen, Joni; Hatsukami, Dorothy K.; Hecht, Stephen S.

    2008-01-01

    Acetaldehyde is one of the most prevalent carcinogens in cigarette smoke. It is also a major metabolite of ethanol and is found widely in the human diet and environment. Acetaldehyde DNA adducts are critical for its carcinogenic properties. The role of acetaldehyde DNA adducts in human cancer related to tobacco and alcohol exposure could be investigated with a suitable biomarker. Therefore, in this study we have developed a method for analysis of the major DNA adduct of acetaldehyde, N2-ethylidene-dGuo (1), in human leukocyte DNA. Leukocyte DNA was subjected to enzyme hydrolysis in the presence of NaBH3CN, which converts adduct 1 to N2-ethyl-dGuo (2). [15N5]N2-ethyl-dGuo was used as internal standard. After solid phase extraction, N2-ethyl-dGuo was quantified by LC-ESI-MS/MS-SRM. The method was sensitive, accurate, and precise, and applicable to low microgram amounts of DNA. It was applied to investigate the effect of smoking cessation on levels of adduct 1, measured as adduct 2. Twenty-five smokers who were only light drinkers were eligible for the study. Levels of adduct 2 were quantified at two baseline time points separated by one week, and again after four weeks of abstinence from smoking and alcohol consumption. The mean (± S.D.) levels of adduct 2 measured in the leukocytes of the smokers were 1310 ± 1720 (range 124 – 7700) and 1120 ± 1140 (range 138 – 5760) fmol/μmol dGuo at the two baseline points and 705 ± 438 (range 111 – 1530) fmol/μmol dGuo after 4 weeks of cessation. The median level of adduct 2 decreased significantly by 28% upon quitting smoking (P = 0.02). These results demonstrate that the major acetaldehyde DNA adduct can be reliably quantified by MS/MS methods in human leukocyte DNA and that cigarette smoking has a modest but significant effect on its levels. PMID:17226933

  10. Metabolism of benzo(a)pyrene by human mammary epithelial cells: toxicity and DNA adduct formation

    SciTech Connect

    Stampfer, M.R.; Batholomew, J.C.; Smith, H.S.; Bartley, J.C.

    1981-10-01

    Pure cultures of human breast epithelial cells and of fibroblastic cells in early passage provided the opportunity to ask whether either cell type had the capability for metabolizing chemical carcinogens and, if so, was the fate of the metabolic products compatible with chemical carcinogens being a factor in the initiation of breast cancer in women. For this purpose, cells were exposed to benzo(a)pyrene(BaP), and (i) the influence on growth potential and (ii) the extent, type, and persistence of adducts between the metabolites of BaP and DNA were measured. Compared with fibroblasts, inhibition of growth by epithelial cells was 50-100 times more sensitive to BaP. Because of this differential sensitivity, epithelial cells were exposed to 0.4 ..mu..M BaP and fibroblasts were exposed to 4.0 ..mu..M BaP in the studies of DNA adduct formation. Separation by high-pressure liquid chromatography of adducts between (+/-)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BaP diol epoxide) and nucleosides from purified DNA revealed that epithelial cells contained modified DNA within 6 hr after adding BaP. Adducts between the 7R anti stereoisomer of BaP diol epoxide and deoxyguanosine predominated at all times. syn BaP diol epoxide adducts with deoxyguanosine and what appeared to be BaP diol epoxide adducts with deoxycytidine were consistently present but at much lower frequency. All three types of BaP diol epoxide-DNA adducts persisted in epithelial cells for 72 hr in BaP-free medium. No adducts were detected in fibroblastic cultures until 96 hr after first exposure to BaP. At this time, the type and extent of BaP diol epoxide-DNA adduct formation was similar to that in epithelial cells exposed to one-tenth the dose of BaP. The type, extent, rate of formation, and persistence of the adducts in human breast epithelial cells was similar to that in cells transformable by exposure to BaP, an indication that they may be targets for chemically induced carcinogenesis.

  11. Mass Spectrometry Detection of Isolevuglandin Adduction to Specific Protein Residues

    PubMed Central

    Charvet, Casey D.; Pikuleva, Irina A.

    2014-01-01

    The aging process seems to be associated with oxidative stress and hence increased production of lipid peroxidation products, including isolevuglandins (isoLGs). The latter are highly reactive γ-ketoaldehydes which can form covalent adducts with primary amino groups of enzymes and proteins and alter the properties of these biomolecules. Yet, little is currently known about amino acid-containing compounds affected by isoLG modification in different age-related pathological processes. To facilitate the detection of these biomolecules, we developed a strategy in which the purified enzyme (or protein) of interest is first treated with authentic isoLG in vitro to evaluate whether it contains reactive lysine residues prone to modification with isoLGs. The data obtained serve as a basis for making the “GO/NO GO” decision as to whether to pursue a further search of this isoLG modification in a biological sample. In this chapter, we describe the conditions for the in vitro isoLG modification assay and how to use mass spectrometry to identify the isoLG-modified peptides and amino acid residues. Our studies were carried out on cytochrome P450 27A1, an important metabolic enzyme, and utilized iso[4]levuglandin E2 as a prototypical isoLG. The isoLG-treated cytochrome P450 was subjected to proteolysis followed by liquid chromatography-tandem mass spectrometry for peptide separation and analysis by Mascot, a proteomics search engine, for the presence of modified peptides. The developed protocol could be applied to characterization of other enzymes/proteins and other types of unconventional post-translational protein modification. PMID:25323515

  12. Inhibition of catechol-O-methyltransferase increases estrogen-DNA adduct formation

    PubMed Central

    Zahid, Muhammad; Saeed, Muhammad; Lu, Fang; Gaikwad, Nilesh; Rogan, Eleanor; Cavalieri, Ercole

    2007-01-01

    The association found between breast cancer development and prolonged exposure to estrogens suggests that this hormone is of etiologic importance in the causation of the disease. Studies on estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA forms depurinating adducts [4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua]. These adducts cause mutations leading to the initiation of breast cancer. Catechol-O-methyltransferase (COMT) is considered an important enzyme that protects cells from the genotoxicity and cytotoxicity of catechol estrogens, by preventing their conversion to quinones. The goal of the present study was to investigate the effect of COMT inhibition on the formation of depurinating estrogen-DNA adducts. Immortalized human breast epithelial MCF-10F cells were treated with 4-OHE2 (0.2 or 0.5 μM) for 24 h at 120, 168, 216, and 264 h post-plating or one time at 1–30 μM 4-OHE2 with or without the presence of COMT inhibitor (Ro41-0960). The culture media were collected at each point, extracted by solid-phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector. The results demonstrate that MCF-10F cells oxidize 4-OHE2 to E1(E2)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. The COMT inhibitor Ro41-0960 blocked the methoxylation of catechol estrogens, with concomitant 3–4 fold increases in the levels of the depurinating adducts. Thus, low activity of COMT leads to higher levels of depurinating estrogen-DNA adducts that can induce mutations and initiate cancer. PMID:17964424

  13. 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

  14. Carcinogen adducts as an indicator for the public health risks of consuming carcinogen-exposed fish and shellfish.

    PubMed Central

    Dunn, B P

    1991-01-01

    A large variety of environmental carcinogens are metabolically activated to electrophilic metabolites that can bind to nucleic acids and protein, forming covalent adducts. The formation of DNA-carcinogen adducts is thought to be a necessary step in the action of most carcinogens. Recently, a variety of new fluorescence, immunochemical, and radioactive-postlabeling procedures have been developed that allow the sensitive measurement of DNA-carcinogen adducts in organisms exposed to environmental carcinogens. In some cases, similar procedures have been developed for protein-carcinogen adducts. In an organism with active metabolic systems for a given carcinogen, adducts are generally much longer lived than the carcinogens that formed them. Thus, the detection of DNA- or protein-carcinogen adducts in aquatic foodstuffs can act as an indicator of prior carcinogen exposure. The presence of DNA adducts would, in addition, suggest a mutagenic/carcinogenic risk to the aquatic organism itself. Vertebrate fish are characterized by high levels of carcinogen metabolism, low body burdens of carcinogen, the formation of carcinogen-macromolecule adducts, and the occurrence of pollution-related tumors. Shellfish, on the other hand, have low levels of carcinogen metabolism, high body burdens of carcinogen, and have little or no evidence of carcinogen-macromolecule adducts or tumors. The consumption of carcinogen adducts in aquatic foodstuffs is unlikely to represent a human health hazard. There are no metabolic pathways by which protein-carcinogen or DNA-carcinogen adducts could reform carcinogens. Incorporation via salvage pathways of preformed nucleoside-carcinogen adducts from foodstuffs into newly synthesized human DNA is theoretically possible.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 1. FIGURE 1. FIGURE 2. PMID:2050048

  15. Carcinogen adducts as an indicator for the public health risks of consuming carcinogen-exposed fish and shellfish

    SciTech Connect

    Dunn, B.P. )

    1991-01-01

    A large variety of environmental carcinogens are metabolically activated to electrophilic metabolites that can bind to nucleic acids and protein, forming covalent adducts. The formation of DNA-carcinogen adducts is thought to be a necessary step in the action of most carcinogens. Recently, a variety of new fluorescence, immunochemical, and radioactive-postlabeling procedures have been developed that allow the sensitive measurement of DNA-carcinogen adducts in organisms exposed to environmental carcinogens. In some cases, similar procedures have been developed for protein-carcinogen adducts. In an organism with active metabolic systems for a given carcinogen, adducts are generally much longer lived than the carcinogens that formed them. Thus, the detection of DNA- or protein-carcinogen adducts in aquatic foodstuffs can act as an indicator of prior carcinogen exposure. The presence of DNA adducts would, in addition, suggest a mutagenic/carcinogenic risk to the aquatic organism itself. Vertebrate fish are characterized by high levels of carcinogen metabolism, low body burdens of carcinogen, the formation of carcinogen-macromolecule adducts, and the occurrence of pollution-related tumors. Shellfish, on the other hand, have low levels of carcinogen metabolism, high body burdens of carcinogen, and have little or no evidence of carcinogen-macromolecule adducts or tumors. The consumption of carcinogen adducts in aquatic foodstuffs is unlikely to represent a human health hazard. There are no metabolic pathways by which protein-carcinogen or DNA-carcinogen adducts could reform carcinogens. Incorporation via salvage pathways of preformed nucleoside-carcinogen adducts from foodstuffs into newly synthesized human DNA is theoretically possible.

  16. Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage.

    PubMed

    Yu, Rui; Lai, Yongquan; Hartwell, Hadley J; Moeller, Benjamin C; Doyle-Eisele, Melanie; Kracko, Dean; Bodnar, Wanda M; Starr, Thomas B; Swenberg, James A

    2015-07-01

    Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N(2-)hydroxymethyl-dG (N(2)-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N(2)-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [(13)CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N(2)-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair.

  17. Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

    PubMed Central

    Yu, Rui; Lai, Yongquan; Hartwell, Hadley J.; Moeller, Benjamin C.; Doyle-Eisele, Melanie; Kracko, Dean; Bodnar, Wanda M.; Starr, Thomas B.; Swenberg, James A.

    2015-01-01

    Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N2-hydroxymethyl-dG (N2-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N2-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [13CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N2-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency’s Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair. PMID:25904104

  18. Simultaneous measurement of benzo[a]pyrene-induced Pig-a and lacZ mutations, micronuclei and DNA adducts in Muta™ Mouse.

    PubMed

    Lemieux, Christine L; Douglas, George R; Gingerich, John; Phonethepswath, Souk; Torous, Dorothea K; Dertinger, Stephen D; Phillips, David H; Arlt, Volker M; White, Paul A

    2011-12-01

    In this study we compared the response of the Pig-a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28-day repeat dose study. Muta™Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig-a mutants. BaP-derived DNA adducts were detected in all tissues examined and significant dose-dependent increases in mutant Pig-a phenotypes (i.e., RET(CD24-) and RBC (CD24-)) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig-a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig-a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose-related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig-a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)-anchor deficiencies detected in the Pig-a assay.

  19. Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL).

    PubMed

    Follmer, Cristian; Coelho-Cerqueira, Eduardo; Yatabe-Franco, Danilo Y; Araujo, Gabriel D T; Pinheiro, Anderson S; Domont, Gilberto B; Eliezer, David

    2015-11-13

    Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons. PMID:26381411

  20. Formation of metal-ion adducts and evidence for surface-catalyzed ionization in electrospray analysis of pharmaceuticals and pesticides

    USGS Publications Warehouse

    Thurman, E.M.; Ferrer, I.

    2002-01-01

    The formation of metal ion adducts in liquid chromatography/mass spectrometry positive-ion electrospray analysis of pharmaceuticals and pesticides was investigated. The evidence of surface-catalyzed ionization in the electrospray analysis was also studied. Both positive and negative ion mass spectrometry were used for the analysis of the products. It was found that the sodium adducts formed in the analysis included single, double, and triple sodium adducts. Adduction was found to occur by attachment of the metal ion to carboxyl, carbonyl and aromatic pi electrons of the molecule.

  1. Cisplatin intrastrand adducts sensitize DNA to base damage by hydrated electrons.

    PubMed

    Behmand, B; Wagner, J R; Sanche, L; Hunting, D J

    2014-05-01

    The oligonucleotide TTTTTGTGTTT with or without a cisplatin adduct was reacted with hydrated electrons generated by ionizing radiation. Hydroxyl radicals were quenched with ethylenediaminetetraacetic acid (EDTA), and the solutions were bubbled with wet nitrogen to eliminate oxygen, a scavenger of hydrated electrons. Prior to irradiation, the structure of the initial cisplatin adduct was identified by mass spectrometry as G-cisplatin-G. Radiation damage to DNA bases was quantified by high-performance liquid chromatography (HPLC), after enzymatic digestion of the TTTTTGTGTTT-cisplatin complex to deoxyribonucleosides. The masses of the platinum adducts following digestion and separation by HPLC were measured by mass spectrometry. Our results demonstrate that hydrated electrons induce damage to thymines as well as detachment of the cisplatin moiety from both guanines in the oligonucleotide. This detachment regenerates both unmodified guanine and damaged guanine, in equimolar amounts. At 1000 Gy, a net average of 2.5 thymines and 1 guanine are damaged for each platinum lost from the oligonucleotide. Given the extensive base damage that occurs for each cisplatin adduct lost, it is clear that, prior to undergoing detachment, these adducts must catalyze several cycles of reactions of hydrated electrons with DNA bases. It is likely that a single reaction leads to the loss of the cisplatin adduct and the damage observed on the guanine base; however, the damage to the thymine bases must require the continued presence of the cisplatin adduct, acting as a catalyst. To our knowledge, this is the first time that platinum-DNA adducts have been shown to have catalytic activity. We propose two pathways for the interaction of hydrated electrons with TTTTTGTGTTT-cisplatin: (1) the hydrated electron is initially captured by a thymine base and transferred by base to base electron hopping to the guanine site, where the cisplatin moiety detaches from the oligonucleotide via dissociative

  2. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    PubMed

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

  3. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    PubMed

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. PMID:24623226

  4. Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps.

    PubMed

    Bézière, Nicolas; Hardy, Micael; Poulhès, Florent; Karoui, Hakim; Tordo, Paul; Ouari, Olivier; Frapart, Yves-Michel; Rockenbauer, Antal; Boucher, Jean-Luc; Mansuy, Daniel; Peyrot, Fabienne

    2014-02-01

    Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H2O2, hydroxyl radical, or peroxynitrite (ONOO(-)). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes. PMID:24161442

  5. Determination of double bond location in fatty acids by manganese adduction and electron induced dissociation.

    PubMed

    Yoo, Hyun Ju; Håkansson, Kristina

    2010-08-15

    Double bond locations in fatty acids can be determined from characteristic charge-remote fragmentation patterns of alkali metal-adducted fatty acids following high energy collision activated dissociation (CAD). With low energy CAD, several chemical derivatization methods, including ozonization, epoxidation, and hydroxylation, have been used to generate characteristic fragments. However, high energy CAD is not universally available and involves a high degree of scattering, causing product ion loss. Further, derivatization reactions involve side reactions and sample loss. Here, we analyzed metal-adducted fatty acids to investigate the utility of electron induced dissociation (EID) for determining double bond location. EID has been proposed to involve both electronic excitation, similar to high energy CAD, and vibrational excitation. Various metals (Li, Zn, Co, Ni, Mg, Ca, Fe, and Mn) were investigated to fix one charge at the carboxylate end of fatty acids to promote charge-remote fragmentation. EID of Mn(II)-adducted fatty acids allowed determination of all double bond locations of arachidonic acid, linolenic acid, oleic acid, and stearic acid. For Mn(II)-adducted fatty acids, reduced characteristic charge-remote product ion abundances at the double bond positions are indicative of double bond locations. However, other metal adducts did not generally provide characteristic product ion abundances at all double bond locations.

  6. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    PubMed

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-01

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties. PMID:27387593

  7. Distribution of ethanol-induced protein adducts in vivo: relationship to tissue injury.

    PubMed

    Niemelä, O

    2001-12-15

    Generation of oxygen free radicals and reactive aldehydes as a result of excessive ethanol consumption has been well established. Recent studies in human alcoholics and in experimental animal models have indicated that acetaldehyde, the first metabolite of ethanol, and the aldehydic products of lipid peroxidation can bind to proteins in tissues forming stable adducts. The demonstration of such adducts in zone 3 hepatocytes in alcoholics with an early phase of histological liver damage indicates that adduct formation may have an important role in the sequence of events leading to alcoholic liver disease. There may be interference with cellular functions, stimulation of fibrogenesis, and immunological responses. Autoantibodies towards distinct types of adducts have been shown to be associated with the severity of liver disease in alcoholic patients. High fat diet and/or iron supplementation combined with ethanol may increase the amount of aldehyde-derived epitopes and promote fibrogenesis in the liver. Recently, ethanol-derived protein modifications have also been found from other tissues exposed to ethanol and acetaldehyde, including rat brain after lifelong ethanol administration, pancreas, and rat muscle. Elevated adduct levels also occur in erythrocytes of alcoholics, which may be related to ethanol-induced morphological aberrations in hematopoiesis.

  8. Nitroreduction and formation of hemoglobin adducts in rats with a human intestinal microflora

    SciTech Connect

    Scheepers, P.T.J.; Straetemans, M.M.E.; Koopman, J.P.; Bos, R.P.

    1994-10-01

    In the covalent binding of nitroarenes to macromolecules, nitroreduction is an important step. The intestinal microflora represents an enormous potential of bacterial nitroreductase activity. As a consequence, the in vivo nitroreduction of orally administerednitroarenes is primarily located in the intestine. In this study, we have investigated the nitroreduction of 2-nitrofluorene (2-NF) by a human microflora in female Wistar rats. Germ-free (FG) rats were equipped with a bacterial flora derived from human feces. Nontreated GF rats and GF animals equipped with a conventional rat flora were used as controls. The composition of the human and the conventional microflora isolated from the rats were consistent with the microflora of the administered feces. In the rats receiving only sunflower seed oil, no adducts were detected. The animals equipped with a human or rat microflora that received 2-aminofluorene (2-AF) formed 2-AF hemoglobin (Hb)-adducts at average levels mean {+-} 0.003 and 0.043 {+-} 0.010 {mu}mole/g Hb, respectively. In the FG rats, an adduct level of 0.57 {+-} 0.09 was determined after 2-AF administration and non adducts were detected after 2-NF administration. The results show that nitroreduction by an acquired human intestinal microflora and subsequent adduct formation can be studied in the rate in vivo. 21 refs., 3 tabs.

  9. Lipoproteins accumulate in immune deposits and are modified by lipid peroxidation in passive Heymann nephritis.

    PubMed Central

    Exner, M.; Susani, M.; Witztum, J. L.; Hovorka, A.; Curtiss, L. K.; Spitzauer, S.; Kerjaschki, D.

    1996-01-01

    Proteinuria in passive Heymann nephritis is primarily caused by reactive oxygen species that are produced by glomerular cells. Reactive oxygen species apparently exert their damaging effects on the glomerular filter by lipid peroxidation and subsequent adduct formation on matrix proteins of glomerular basement membranes. This raised the question as to the source of polyunsaturated fatty acids required as substrates for lipid peroxidation. Here we have localized by immunocytochemistry rat apolipoprotein E and apolipoprotein B within subepithelial immune deposits. Moreover, apolipoprotein B extracted from isolated glomeruli of proteinuric passive Heymann nephritis rats shows degradation and lipid peroxidation adduct formation, similar to apoproteins of oxidized lipoproteins in atherosclerotic lesions. These data provide evidence that lipoproteins accumulate within immune deposits and suggest that their lipids generate lipid-peroxidation-derived reactive compounds. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8863678

  10. The metabolic activation and DNA adducts of dinitropyrenes.

    PubMed

    Beland, F A

    1986-08-01

    Dinitropyrenes are contaminants in diesel emissions that are mutagenic in bacteria and mammalian cells, and tumorigenic in laboratory animals. In this project, we investigated the factors that contributed to the extreme genotoxicity of dinitropyrenes in bacteria and determined if these factors were important in mammalian cells. Xanthine oxidase, a mammalian nitroreductase, catalyzed the conversion of the dinitropyrenes to DNA-bound products, but the level of binding did not exceed that observed with 1-nitropyrene. This suggested that factors in addition to nitroreduction were important in the metabolic activation of dinitropyrenes. 1-Nitro-6-nitrosopyrene and 1-nitro-8-nitrosopyrene were synthesized and reacted with DNA under reducing conditions. The same C8-substituted deoxyguanosine adducts were formed that were found in the xanthine oxidase-catalyzed reactions, which confirmed that incubation with this nitroreductase generated reactive N-hydroxy arylamine intermediates. In incubations with rat and human liver microsomes and cytosol, 1-nitropyrene and 1,3-dinitropyrene were reduced to a lesser extent than 1,6- and 1,8-dinitropyrene, which was in accord with their relative mutagenicities. Each of the cytosolic incubations were similar in that oxygen decreased aminopyrene, but not nitrosopyrene, formation. The data indicated that reduced derivatives of the nitrosopyrenes were redox cycling with oxygen, which decreased cytosolic aminopyrene formation. In cytosolic incubations, oxygen inhibited the reduction of 1-nitropyrene and 1,3-dinitropyrene to a greater extent than 1,6- and 1,8-dinitropyrene. By comparison, in microsomal investigations, the nitroreduction of each nitrated pyrene was equally oxygen-sensitive. This apparently was caused by the initial nitroanion radicals reacting with oxygen to decrease nitrosopyrene formation. Although more extensive nitroreduction of each compound was detected in anaerobic incubations, aerobic reduction of these compounds did

  11. Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon black and titanium dioxide particles.

    PubMed

    Gallagher, J; Heinrich, U; George, M; Hendee, L; Phillips, D H; Lewtas, J

    1994-07-01

    Exposure of rats to diesel emissions results in the development of lung tumors. The objective of this study was to determine whether the polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs or other polycyclic organic matter adsorbed to diesel particles induces the formation of DNA adducts in the lung when compared to particles with little or no adsorbed organic matter. Rats were exposed to diesel emissions containing particles with over 30% solvent-extractable adsorbed organic matter and to particles with < 0.1% adsorbed organic matter (carbon black particles and TiO2). Wistar rats were exposed to diesel emissions (7.5 mg/m3) for 2 months, 6 months and 2 years and for 2 years to carbon black (11.3 mg/m3) and TiO2 particles (10.4 mg/m3) to compare tumorigenic response and DNA adduct formation in the lung. Two versions of the 32P-postlabeling assay for the detection of DNA adducts were used to tentatively identify nitrated-amine or arylamine adducts formed relative to other nitro PAH based on the demonstrated sensitivity of these adducts to nuclease P1 treatment. Total adduct levels were determined for peripheral lung tissue DNA as detected in a diagonal radioactive zone. One major adduct which migrated outside this region (adduct 1) and a nuclease P1-sensitive adduct (adduct 2) were quantitated separately. Adduct 1 increased significantly over time in the filtered air exposed animals but decreased markedly at the 2 year time points regardless of particle type, presumably as a result of adduct dilution through de novo cell synthesis or cell proliferation invoked in response to particle loading and/or effect on the endogenous synthesis or degradation of DNA reactive moieties. The nuclease sensitive adduct (adduct 2), possibly resulting from exposure to nitro-PAHs, was detected in diesel-exposed rats but was not detected in the rats exposed to TiO2 and carbon black. No significant elevation in PAH-derived adducts, relative to the filtered air controls, was observed in

  12. Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon black and titanium dioxide particles.

    PubMed

    Gallagher, J; Heinrich, U; George, M; Hendee, L; Phillips, D H; Lewtas, J

    1994-07-01

    Exposure of rats to diesel emissions results in the development of lung tumors. The objective of this study was to determine whether the polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs or other polycyclic organic matter adsorbed to diesel particles induces the formation of DNA adducts in the lung when compared to particles with little or no adsorbed organic matter. Rats were exposed to diesel emissions containing particles with over 30% solvent-extractable adsorbed organic matter and to particles with < 0.1% adsorbed organic matter (carbon black particles and TiO2). Wistar rats were exposed to diesel emissions (7.5 mg/m3) for 2 months, 6 months and 2 years and for 2 years to carbon black (11.3 mg/m3) and TiO2 particles (10.4 mg/m3) to compare tumorigenic response and DNA adduct formation in the lung. Two versions of the 32P-postlabeling assay for the detection of DNA adducts were used to tentatively identify nitrated-amine or arylamine adducts formed relative to other nitro PAH based on the demonstrated sensitivity of these adducts to nuclease P1 treatment. Total adduct levels were determined for peripheral lung tissue DNA as detected in a diagonal radioactive zone. One major adduct which migrated outside this region (adduct 1) and a nuclease P1-sensitive adduct (adduct 2) were quantitated separately. Adduct 1 increased significantly over time in the filtered air exposed animals but decreased markedly at the 2 year time points regardless of particle type, presumably as a result of adduct dilution through de novo cell synthesis or cell proliferation invoked in response to particle loading and/or effect on the endogenous synthesis or degradation of DNA reactive moieties. The nuclease sensitive adduct (adduct 2), possibly resulting from exposure to nitro-PAHs, was detected in diesel-exposed rats but was not detected in the rats exposed to TiO2 and carbon black. No significant elevation in PAH-derived adducts, relative to the filtered air controls, was observed in

  13. Formation and persistence of DNA adducts during and after a long-term administration of 2-nitrofluorene.

    PubMed

    Cui, X S; Eriksson, L C; Möller, L

    1999-06-01

    2-Nitrofluorene (NF) is an environmental pollutant. Our previous studies have shown that NF is a carcinogen, primarily targeting the liver, kidney and forestomach in rats. NF-induced DNA adducts were also shown higher levels in the tumor-targeting tissues compared to non-tumor targeting organs. The present study was aimed to observe the kinetics of DNA adduct formation and persistence during the process of NF-induced tumor formation. NF was supplemented in diet at three dose levels and was fed to rats continuously for up to 11 months. DNA adduct formation in the liver, kidney, spleen and stomach of rats after different period (10 days and 11 months) of NF administration was analyzed with 32P-HPLC techniques. DNA adduct persistence in the liver was also assessed after the withdrawal of NF administration. Four major NF-DNA adducts (adducts A, B, C and D) were found in the liver and kidney. DNA adduct D showed high level in the forestomach mucosa after 10 days of NF feeding while adducts A and C were undetectable. DNA adduct C and D co-migrated with C3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), respectively, by 32P-HPLC co-chromatography. DNA adducts A and B constituted the major part (>80%) of NF-DNA adducts after a long period (11 months) of NF feeding. The four NF-DNA adducts showed different recovery from different enrichment procedures, i.e., nuclease P1 or butanol treatment. Three out of the four NF-DNA adducts were still detectable in the rat liver after 11 months on the basal diet. In conclusion, four major DNA adducts are induced by NF oral administration. Among those, one is identified as dG-N2-AAF and another one as dG-C8-AF. The four NF-DNA adducts showed different kinetics of formation and persistence, which may play different roles in NF-induced tumor formation. PMID:10366768

  14. 32P-postlabelling analysis of dibenz[a,j]acridine-DNA adducts in mice: identification of proximate metabolites.

    PubMed

    Talaska, G; Roh, J; Schamer, M; Reilman, R; Xue, W; Warshawsky, D

    1995-03-30

    N-Heterocyclic polynuclear aromatics are widely-occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. Dibenz[a,j]acridine (DBA), a member of this class, has been shown to be a skin carcinogen in mice. We undertook studies to determine the organ distribution of DBA-DNA adducts and to identify the DBA metabolites which lead to the formation of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD) trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD) and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were topically applied on mice. DNA was isolated using enzyme-solvent extraction methods, and analyzed for carcinogen-DNA adducts using 32P-postlabelling. In skin, DBA produced two distinct adducts (Adducts 1 and 2). The same two adducts were seen when DBA-3,4-DHD was applied. In addition, the total adduct level elicited by DBA-3,4-DHD was twice that of the parent compound. Two adducts (Adducts 3 and 4) were also seen in mouse skin when DBA-5,6-DHD was applied, but these differed chromatographically from adducts seen with DBA. However, when DBA-3,4-DHD was applied and analyzed using sensitive nuclease P1 32P-postlabelling, all four adducts could be detected. These results suggest that the major route of DBA activation to DNA-binding species in skin is through formation of DBA-3,4-DHD and subsequent metabolism of this compound to a bay-region diol-epoxide. However, we postulate that another activation pathway may proceed through a bis-dihydrodiol-epoxide.

  15. Inhibition of Norovirus 3CL Protease by Bisulfite Adducts of Transition State Inhibitors

    PubMed Central

    Mandadapu, Sivakoteswara Rao; Gunnam, Mallikarjuna Reddy; Tiew, Kok-Chuan; Uy, Roxanne Adeline Z.; Prior, Allan M.; Alliston, Kevin R.; Hua, Duy H.; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C.

    2013-01-01

    Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the U.S. alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED50 of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters. PMID:23218713

  16. Reactivity of adducts relevant to the deposition of hexagonal BN from first-principles calculations

    NASA Astrophysics Data System (ADS)

    Freitas, R. R. Q.; Gueorguiev, G. K.; de Brito Mota, F.; de Castilho, C. M. C.; Stafström, S.; Kakanakova-Georgieva, A.

    2013-09-01

    First-principles calculations, which also implement the nudged elastic band (NEB) code, are performed to investigate (i) the stability of the (C2H5)3B:NH3 adduct formed by the initial precursor molecules triethylborane (C2H5)3B and ammonia NH3 in the metal-chemical-vapor-deposition (MOCVD) of hexagonal BN, and (ii) the energy barrier to the first ethane elimination through consistent unimolecular, ammonia-assisted, and adduct-assisted reaction pathways. Comparison is done with the reference case of the (CH3)3Al:NH3 adduct, notoriously known for its high degree of stability and reactivity, which determines an overall severe parasitic gas-phase chemical reaction mechanism in the deposition of AlN.

  17. Comparison of MS/MS Methods for Characterization of DNA/cisplatin Adducts

    PubMed Central

    Xu, Zhe; Shaw, Jared B.; Brodbelt, Jennifer S.

    2012-01-01

    The development of activation/dissociation techniques such as ultraviolet photodissociation (UVPD), infrared multiphoton dissociation (IRMPD) and electron transfer dissociation (ETD) as alternatives to collision induced dissociation (CID) has extended the range of strategies for characterizing biologically relevant molecules. Here, we describe a comprehensive comparison of CID, IRMPD, UVPD, ETD and hybrid processes termed ETcaD and ET-IRMPD (and analogous hybrid methods in the negative mode NETcaD and NET-IRMPD) for generating sequence specific-fragment ions and allowing adduction sites to be pinpointed for DNA/cisplatin adducts. Among the six MS/MS methods, the numerous products generated by the IRMPD and UVPD techniques resulted in the most specific and extensive backbone cleavages. We conclude that IRMPD and UVPD methods generally offer the best characteristics for pinpointing the cisplatin adduction sites in the fragment-rich spectra. PMID:23264150

  18. Rotational Investigation of the Adducts of Formic Acid with Alcohols, Ethers and Esters

    NASA Astrophysics Data System (ADS)

    Evangelisti, Luca; Spada, Lorenzo; Li, Weixing; Caminati, Walther

    2016-06-01

    Mixtures of formic acid with methyl alcohol, with isopropyl alcohol, with tert-butyl alcohol, with dimethylether and with isopropylformiate have been supersonically expanded as pulsed jets. The obtained cool plumes have been analyzed by Fourier transform microwave spectroscopy. It has been possible to assign the rotational spectra of the 1:1 adducts of formic acid with tert-butyl alcohol, with dimethyl ether and with isopropylformiate. The conformational shapes and geometries of these adducts, as well as the topologies of their itermolecular hydrogen bonds will be presented. An explanation is given of the failure of the assignments of the rotational spectra of the adducts of formic acid with methyl alcohol and isopropyl alcohol.

  19. The influence of antagonist muscle electrical stimulation on maximal hip adduction force

    PubMed Central

    Nakano, Sota; Wada, Chikamune

    2016-01-01

    [Purpose] The aim of this study was to determine whether electrical stimulation of the tensor fascia lata muscle decreases voluntary maximum resistance to passive abduction motion in participants without disease of the central nervous system. [Subjects] The participants were 16 healthy men. [Methods] The hip joint was moved from 10° adduction to 0° adduction with an angular velocity of 7°/s. During the passive leg motion, the subject was asked to resist the motion with maximum force. Two experimental conditions were prepared: (1) electrical stimulation provided to the tensor fascia lata muscle during the passive motion; and (2) no electrical stimulation provided. [Results] The force was 10.2 ± 3.5 kgf with electrical stimulation and 12.2 ± 3.8 kgf without electrical stimulation. [Conclusion] The results suggested that the maximum hip adduction force decreased in participants because of electrical stimulation of the tensor fascia lata muscle. PMID:26957742

  20. Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors.

    PubMed

    Mandadapu, Sivakoteswara Rao; Gunnam, Mallikarjuna Reddy; Tiew, Kok-Chuan; Uy, Roxanne Adeline Z; Prior, Allan M; Alliston, Kevin R; Hua, Duy H; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C

    2013-01-01

    Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.

  1. Potential application of benzo(a)pyrene-associated adducts (globin or lipid) as blood biomarkers for target organ exposure and human risk assessment.

    PubMed

    Kwack, Seung Jun; Kim, Dae Young; Kim, Yeon Joo; Roh, Tae Hyun; Choi, Seul Min; Lim, Duck Soo; Shin, Han-Seung; Kim, Hyung Sik; Lee, Byung-Mu

    2014-01-01

    In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen-adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [(3)H]BaP into female ICR mice for 7 d. Following treatment with [(3)H]BaP, formation of [(3)H]BaP-DNA or -protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [(3)H]BaP indicated that BaP-globin adduct formation and BaP-lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP-globin adduct formation or BaP-lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure. PMID:25343297

  2. "Danger" conditions increase sulfamethoxazole-protein adduct formation in human antigen-presenting cells.

    PubMed

    Lavergne, S N; Wang, H; Callan, H E; Park, B K; Naisbitt, D J

    2009-11-01

    Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such "danger signals" on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 microM-2 mM; 5 min-24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1beta, IL-6, IL-10; tumor necrosis factor-alpha; interferon-gamma; and transforming growth factor-beta], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H(2)O(2)), and hyperthermia (37.5-39.5 degrees C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant

  3. Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.

    PubMed

    Vaidyanathan, Vaidyanathan G; Liang, Fengting; Beard, William A; Shock, David D; Wilson, Samuel H; Cho, Bongsup P

    2013-08-01

    The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase β (pol β) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

  4. Conformational evaluation of DNA-carcinogen adducts using semi-empirical potential energy calculations

    SciTech Connect

    Verna, L.K.

    1992-01-01

    The covalent attachment of an aromatic amine to guanine C8 can produce a conformational change within the DNA molecule. This conformational change is likely to influence the altered DNA's biological capacity. The author used semi-empirical potential energy calculations to evaluate conformational patterns of DNA-aromatic amine adducts using the series: aniline, 4-aminobiphenyl, 2-aminofluorene and 1-aminopyrene. An exhaustive search was made of the conformational space for carcinogen modified two-base sequences. Information was incorporated into single stranded modified trimers. Modified strands were incorporated in duplex trimers. Nine-base modified duplexes were constructed and evaluated. This procedure produced distinctly different patterns for each aromatic amine investigated. It was apparent that the base sequence in which the carcinogen modification was found was crucial to the conformational change produced. At the dimer level, aniline allows both syn and anti guanine orientations at the carcinogen modification site. There were base-base and base-carcinogen stacked states, suggesting a flexible adduct easily able to assume many conformations. 4-Aminobiphenyl attachment resulted in low energy base-carcinogen stacked states, and a guanine torsion predominantly in a low syn orientation. The flexibility of this adduct was greatly reduced from that of the aniline adduct. 2-Aminofluorene adducts assumed more of a conformational mix. The major portion was base-base stacked with modified guanine anti, with a portion with base-carcinogen stacking and guanine syn or low syn. 1-Aminopyrene adducts were inflexible. The majority assumed a base-carcinogen stack with guanine syn. The conformational profiles of large modified pieces provided details of a unique low energy wedge conformation, in which aminofluorene, particularly, was able to fit into the minor groove with very little helix distortion.

  5. Safrole-DNA adduct in hepatocellular carcinoma associated with betel quid chewing.

    PubMed

    Chung, Yu-Ting; Chen, Chiu-Lan; Wu, Cheng-Chung; Chan, Shan-An; Chi, Chin-Wen; Liu, Tsung-Yun

    2008-12-15

    Betel quid chewing, which contributes high concentration of safrole in saliva, is a popular oral habit in Taiwan. Safrole is a documented rodent hepatocarcinogen, yet its hepatocarcinogenic potential in human is not known. Here, we used LC/ESI-ITMS(n) and LC/QTOF-MS confirmed safrole-dGMP as reference standard to detect the safrole-DNA adduct in hepatic tissues from HBsAg-/HCV-seronegative hepatocellular carcinoma patients by (32)P-postlabeling. We first synthesized and confirmed safrole-dGMP by LC/MS. Two isomeric safrole-dGMPs were characterized as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine and N(2)-(safrol-1'-yl) deoxyguanosine. This technique was able to detect hepatic safrole-DNA adduct in mice that were treated with safrole but not sensitive enough to detect safrole-DNA adduct in human samples. Using the nuclease P1 version of the (32)P-postlabeling technique, we detected the presence of safrole-DNA adduct in two out of 28 hepatic tissues from hepatocellular carcinoma patients, and only these two patients had a history of betel quid chewing lasting more than 10 years. From co-chromatography with the mass confirmed safrole-dGMPs, this safrole-DNA adduct was identified as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine. These results suggest that betel quid-containing safrole might be involved in the pathogenesis of hepatocellular carcinoma in human beings and LC/MS has the potential to identify DNA adducts in clinical samples.

  6. Diet, metabolic polymorphisms and dna adducts: the EPIC-Italy cross-sectional study.

    PubMed

    Palli, D; Vineis, P; Russo, A; Berrino, F; Krogh, V; Masala, G; Munnia, A; Panico, S; Taioli, E; Tumino, R; Garte, S; Peluso, M

    2000-08-01

    DNA adducts in peripheral leukocytes are considered a reliable indicator of internal dose exposure to genotoxic agents and, possibly, of cancer risk. We investigated their association with diet and other individual characteristics in healthy adults. The prospective study EPIC-Italy, a section of a larger European project, enrolled 47,749 men and women, aged 35-64 years, in 5 centres: all provided individual information about dietary and life-style habits and a blood sample. In a cross-sectional study, approximately 100 volunteers were randomly selected from each of the three main geographical study areas (Northern, Central and Southern Italy). DNA adducts and four polymorphic metabolic genotypes were determined in peripheral leukocytes by using (32)P-postlabelling technique and PCR methods. Among 309 subjects (153 men), 72.8% had detectable levels of DNA adducts (mean: 8.1 +/- 0.6 per 10(9) nucleotides). Strong negative associations emerged with the reported frequency of consumption of fresh fruit and vegetables, olive oil, and the intake of antioxidants. DNA adducts were higher in subjects with GSTT1 null genotype (p = 0.05). Significant differences between study centres emerged in multivariate analyses (mean levels: 11.0, 10.0, 7.2, 6.5 and 5.2 for Florence, Naples, Turin, Varese and Ragusa, respectively). A possible opposite seasonal variation was found according to latitude: adduct levels tended to be lower in winter in Florence and the southern centres, and during summer in the two northern centres. Frequent consumption of fresh fruit and vegetables is associated with reduced levels of DNA adducts, possibly contributing to the role of diet in modulating cancer risk. PMID:10897053

  7. Diet, metabolic polymorphisms and dna adducts: the EPIC-Italy cross-sectional study.

    PubMed

    Palli, D; Vineis, P; Russo, A; Berrino, F; Krogh, V; Masala, G; Munnia, A; Panico, S; Taioli, E; Tumino, R; Garte, S; Peluso, M

    2000-08-01

    DNA adducts in peripheral leukocytes are considered a reliable indicator of internal dose exposure to genotoxic agents and, possibly, of cancer risk. We investigated their association with diet and other individual characteristics in healthy adults. The prospective study EPIC-Italy, a section of a larger European project, enrolled 47,749 men and women, aged 35-64 years, in 5 centres: all provided individual information about dietary and life-style habits and a blood sample. In a cross-sectional study, approximately 100 volunteers were randomly selected from each of the three main geographical study areas (Northern, Central and Southern Italy). DNA adducts and four polymorphic metabolic genotypes were determined in peripheral leukocytes by using (32)P-postlabelling technique and PCR methods. Among 309 subjects (153 men), 72.8% had detectable levels of DNA adducts (mean: 8.1 +/- 0.6 per 10(9) nucleotides). Strong negative associations emerged with the reported frequency of consumption of fresh fruit and vegetables, olive oil, and the intake of antioxidants. DNA adducts were higher in subjects with GSTT1 null genotype (p = 0.05). Significant differences between study centres emerged in multivariate analyses (mean levels: 11.0, 10.0, 7.2, 6.5 and 5.2 for Florence, Naples, Turin, Varese and Ragusa, respectively). A possible opposite seasonal variation was found according to latitude: adduct levels tended to be lower in winter in Florence and the southern centres, and during summer in the two northern centres. Frequent consumption of fresh fruit and vegetables is associated with reduced levels of DNA adducts, possibly contributing to the role of diet in modulating cancer risk.

  8. Comments on the purported generation of formaldehyde and adduct formation from the sweetener aspartame.

    PubMed

    Tephly, T R

    1999-01-01

    A recent paper by Trocho et al. (1) describes experiments meant to show that formaldehyde adducts are formed when rats are administered the sweetener aspartame. These authors assume that the methanol carbon of aspartame generates formaldehyde which then forms adducts with protein, DNA, and RNA. Doses employed range widely. In this letter, studies which have been published previously and which were not cited by these authors are reviewed in order to put into perspective the disposition of methanol and formaldehyde in monkeys and humans, species relevant to the toxicity of methanol and its toxic metabolite, formic acid.

  9. INVESTIGATION OF THE RADICAL-MEDIATED PRODUCTION OF BENZENE OXIDE PROTEIN ADDUCTS IN VITRO AND IN VIVO

    EPA Science Inventory

    High background levels of benzene oxide (BO) adducts with hemoglobin and albumin (BO-Hb and BO-Alb) have been measured in unexposed humans and animals. To test the influence of radical-mediated pathways on production of these BO-protein adducts, we employed Fenton chemistry to...

  10. BINDING OF CARCINOGENS TO DNA AND COVALENT ADDUCTS DNA DAMAGE - PAH, AROMATIC AMINES, NITRO-AROMATIC COMPOUNDS, AND HALOGENATED COMPOUNDS

    EPA Science Inventory

    DNA adducts are the covalent addition products resulting from binding of reactive chemical species to DNA bases. The cancer initiating role of DNA adducts is well-established, and is clearly reflected in the high cancer incidence observed in individuals with deficiencies in any o...

  11. Immunohistochemical localization and quantification of the 3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity.

    PubMed Central

    Roberts, D. W.; Bucci, T. J.; Benson, R. W.; Warbritton, A. R.; McRae, T. A.; Pumford, N. R.; Hinson, J. A.

    1991-01-01

    Acetaminophen overdose causes severe hepatotoxicity in humans and laboratory animals, presumably by metabolism to N-acetyl-p-benzoquinone imine: and binding to cysteine groups as 3-(cystein-S-yl)acetaminophen-protein adduct. Antiserum specific for the adduct was used immunohistochemically to demonstrate the formation, distribution, and concentration of this specific adduct in livers of treated mice and was correlated with cell injury as a function of dose and time. Within the liver lobule, immunohistochemically demonstrable adduct occurred in a temporally progressive, central-to-peripheral pattern. There was concordance between immunohistochemical staining and quantification of the adduct in hepatic 10,000g supernate, using a quantitative particle concentration fluorescence immunoassay. Findings include: 1) immunochemically detectable adduct before the appearance of centrilobular necrosis, 2) distinctive lobular zones of adduct localization with subsequent depletion during the progression of toxicity, 3) drug-protein binding in hepatocytes at subhepatotoxic doses and before depletion of total hepatic glutathione, 4) immunohistochemical evidence of drug binding in the nucleus, and 5) adduct in metabolically active and dividing hepatocytes and in macrophagelike cells in the regenerating liver. Images Figure 2 Figure 4 PMID:1992763

  12. Diagnostic ions for the analysis of phenylalanine adducts of acrylamide and styrene by ESI-QTOF mass spectrometry.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan Antranik

    2013-10-30

    To facilitate the detection of acrylamide or styrene adduct of amino acids by mass spectrometry based techniques, phenylalanine was used as a representative amino acid and pyrolysis was employed in conjunction with isotope labeling technique as a microscale sample preparation tool to generate the reaction products. The residues remaining after the pyrolysis of phenylalanine/styrene, phenylalanine/acrylamide, and phenylalanine/glucose mixtures at 250 °C were analyzed by electrospray quadrupole time-of-flight (ESI-QqTOF) mass spectrometry to identify the adducts. The phenylalanine/acrylamide adduct was independently synthesized for confirmation. Characteristic product ions in the tandem mass spectra were found at m/z 191 for the acrylamide adduct and at m/z 262 and 190 for its double-addition product. On the other hand, an ion at m/z 224 was shown to be diagnostic of the styrene adduct. The ability of the m/z 224 ion to predict the presence of styrene adduct in a heated phenylalanine/glucose model system was tested and verified. Detailed isotope labeling analysis of the phenylalanine/glucose model further indicated the formation of a novel adduct that was consistent with the reaction of the Amadori product with styrene. Such diagnostic ions that are needed to develop MS/MS-based screening methodologies may accelerate in the future the detection of Michael-type adducts in food.

  13. HNE catabolism and formation of HNE adducts are modulated by beta oxidation of fatty acids in the isolated rat heart

    PubMed Central

    Li, Qingling; Sadhukhan, Sushabhan; Berthiaume, Jessica M.; Ibarra, Rafael A.; Tang, Hui; Deng, Shuang; Hamilton, Eric; Nagy, Laura E.; Tochtrop, Gregory P.; Zhang, Guo-Fang

    2013-01-01

    We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2-(E)-nonenal (HNE) via two parallel pathways, which rely heavily on β oxidation pathways. The hypothesis driving the present report is that perturbations of β oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE modified proteins that may further exacerbate pathology. The current study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1 mM octanoate). During ischemia, HNE concentrations doubled and the glutathione-HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained, however, further catabolism through β oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to β oxidation, such as myocardial ischemia, obesity and high fat diet induced diseases. PMID:23328733

  14. DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

    PubMed Central

    Ikeda, Mio; Furukohri, Asako; Philippin, Gaelle; Loechler, Edward; Akiyama, Masahiro Tatsumi; Katayama, Tsutomu; Fuchs, Robert P.; Maki, Hisaji

    2014-01-01

    Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N2-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N2-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption. PMID:24957605

  15. Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of bis-(2-Chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

    PubMed Central

    Gruppi, Francesca; Hejazi, Leila; Christov, Plamen P.; Krishnamachari, Sesha; Turesky, Robert J.; Rizzo, Carmelo J.

    2015-01-01

    A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS3) method was established to characterize and measure five deoxyguanosine (dG) adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis-(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM-G) adduct and its crosslink (G-NM-G), the ring-opened formamidopyrimidine (FapyG) mono-adduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 μM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 107 DNA bases, when the equivalent of 5 μg DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G mono-adduct; the FapyG-NM-FapyG adduct was at the limit of detection. The NM-FapyG adducts formed in CT DNA at a level of ~20% that of the NM-G adduct. NM-FapyG has not been previously quanitified and the FapyG-NM-G and FapyG-NM-FapyG adducts have not be previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 μM) for 24 h. The major adduct formed was NM-G (970 adducts per 107 bases), followed by G-NM-G (240 adducts per 107 bases) and NM-FapyG (180 adducts per 107 bases), and lastly the FapyG-NM-G cross-link adduct (6.0 adducts per 107 bases). These lesions are expected to contribute to the NM-mediated toxicity and

  16. High resolution-sensitivity characterization of polycyclic aromatic hydrocarbon-DNA adducts using fluorescence line narrowing spectrometry

    SciTech Connect

    Cooper, R.S.

    1988-07-01

    The application of fluorescence line narrowing spectrometry (FLNS) to the investigation of polar polycyclic aromatic hydrocarbon (PAH) metabolites and their corresponding DNA adducts is demonstrated. The selectivity is shown through the successful resolution of all components in separate mixtures of similar but distinct derivatives of benzo(a)pyrene, benz(a)anthracene, and chrysene. The separate mixtures were composed of six metabolites, five DNA adducts, each metabolite and its corresponding DNA adduct, and six metabolites and two DNA adducts. The broad applicability of FLNS is demonstrated through applications to the analysis of globin adducts, PAH metabolites in urine, and real samples, and to the investigation of carcinogenic metabolic pathways. 98 refs., 31 figs., 5 tabs.

  17. Cigarette Smoking, BPDE-DNA Adducts, and Aberrant Promoter Methylations of Tumor Suppressor Genes (TSGs) in NSCLC from Chinese Population.

    PubMed

    Jin, Yongtang; Xu, Peiwei; Liu, Xinneng; Zhang, Chunye; Tan, Cong; Chen, Chunmei; Sun, Xiaoyu; Xu, Yingchun

    2016-01-01

    Non-small cell lung cancer (NSCLC) is related to the genetic and epigenetic factors. The goal of this study was to determine association of cigarette smoking and BPDE-DNA adducts with promoter methylations of several genes in NSCLC. Methylation of the promoters of p16, RARβ, DAPK, MGMT, and TIMP-3 genes of tumor tissues from 199 lung cancer patients was analyzed with methylation-specific PCR (MSP), and BPDE-DNA adduct level in lung cancer tissue was obtained by ELISA. Level of BPDE-DNA adduct increased significantly in males, aged people (over 60 years), and smokers; however, no significant difference was found while comparing the BPDE-DNA adduct levels among different tumor types, locations, and stages. Cigarette smoking was also associated with increased BPDE-DNA adducts level (OR = 2.43, p > .05) and increased methylation level in at least 1 gene (OR = 5.22, p < .01), both in dose-response manner. Similarly, cigarette smoking also significantly increase the risk of p16 or DAPK methylation (OR = 3.02, p < .05 for p16, and 3.66, p < .05 for DAPK). The highest risk of BPDE-DNA adducts was detected among individuals with cigarette smoking for more than 40 pack-years (OR = 4.21, p < .01). Furthermore, the present study did not show that BPDE-DNA adducts are significantly associated with abnormal TSGs methylations in NSCLC, including SCC and AdO, respectively. Conclusively, cigarette smoking is significantly associated with the increase of BPDE-DNA adduct level, promoter hypermethylation of p16 and DAPK genes, while BPDE-DNA adduct was not significantly related to abnormal promoter hypermethylation in TSGs, suggesting that BPDE-DNA adducts and TSGs methylations play independent roles in NSCLC.

  18. Cigarette Smoking, BPDE-DNA Adducts, and Aberrant Promoter Methylations of Tumor Suppressor Genes (TSGs) in NSCLC from Chinese Population.

    PubMed

    Jin, Yongtang; Xu, Peiwei; Liu, Xinneng; Zhang, Chunye; Tan, Cong; Chen, Chunmei; Sun, Xiaoyu; Xu, Yingchun

    2016-01-01

    Non-small cell lung cancer (NSCLC) is related to the genetic and epigenetic factors. The goal of this study was to determine association of cigarette smoking and BPDE-DNA adducts with promoter methylations of several genes in NSCLC. Methylation of the promoters of p16, RARβ, DAPK, MGMT, and TIMP-3 genes of tumor tissues from 199 lung cancer patients was analyzed with methylation-specific PCR (MSP), and BPDE-DNA adduct level in lung cancer tissue was obtained by ELISA. Level of BPDE-DNA adduct increased significantly in males, aged people (over 60 years), and smokers; however, no significant difference was found while comparing the BPDE-DNA adduct levels among different tumor types, locations, and stages. Cigarette smoking was also associated with increased BPDE-DNA adducts level (OR = 2.43, p > .05) and increased methylation level in at least 1 gene (OR = 5.22, p < .01), both in dose-response manner. Similarly, cigarette smoking also significantly increase the risk of p16 or DAPK methylation (OR = 3.02, p < .05 for p16, and 3.66, p < .05 for DAPK). The highest risk of BPDE-DNA adducts was detected among individuals with cigarette smoking for more than 40 pack-years (OR = 4.21, p < .01). Furthermore, the present study did not show that BPDE-DNA adducts are significantly associated with abnormal TSGs methylations in NSCLC, including SCC and AdO, respectively. Conclusively, cigarette smoking is significantly associated with the increase of BPDE-DNA adduct level, promoter hypermethylation of p16 and DAPK genes, while BPDE-DNA adduct was not significantly related to abnormal promoter hypermethylation in TSGs, suggesting that BPDE-DNA adducts and TSGs methylations play independent roles in NSCLC. PMID:27042875

  19. An Adenine-DNA Adduct Derived from Nitroreduction of 6-Nitrochrysene is more Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts

    PubMed Central

    Krzeminski, Jacek; Kropachev, Konstantin; Reeves, Dara; Kolbanovskiy, Aleksandr; Kolbanovskiy, Marina; Chen, Kun-Ming; Sharma, Arun K.; Geacintov, Nicholas; Amin, Shantu; El-Bayoumy, Karam

    2013-01-01

    Previous studies in rats, mice and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: 1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts: N-(dG-8-yl)-6-AC, 5-(dG-N2-yl)-6-AC and N-(dA-8-yl)-6-AC, and 2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield: N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N2-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ~2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ~ 8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N2-yl)-6-AC ~ N-(dG-8-yl)-6-AC ~ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N2-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue, could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland. PMID:24112095

  20. Neurodegeneration with Brain Iron Accumulation

    MedlinePlus

    ... Diversity Find People About NINDS NINDS Neurodegeneration with Brain Iron Accumulation Information Page Synonym(s): Hallervorden-Spatz Disease, ... done? Clinical Trials Organizations What is Neurodegeneration with Brain Iron Accumulation? Neurodegeneration with brain iron accumulation (NBIA) ...

  1. Crystal structure of ball-milled mixture of sodium chloride and magnesium chloride-ethanol adduct

    SciTech Connect

    Jiang Xue; Tian Xiuzhi; Fan Zhiqiang

    2008-02-05

    NaCl doped MgCl{sub 2}.nEtOH adducts were prepared by ball-milling MgCl{sub 2}.2.5EtOH with NaCl. Both the ball-milled MgCl{sub 2}.nEtOH/NaCl mixture and pure MgCl{sub 2}.2.5EtOH adducts were analyzed by X-ray diffraction (XRD), transmission electron microscope (TEM), thermogravimetry (TG) and differencial scanning calorimetry (DSC). A simple MgCl{sub 2}.nEtOH/NaCl mixture without ball-milling treatment was also studied for comparison. Two kinds of mixed crystals, Na{sub 2}MgCl{sub 4} and NaMgCl{sub 3}, were found to be formed in a ball-milled mixture that contained 16 mol.% NaCl. TG and DSC analysis of the samples also provided indirect evidences supporting the presence of the mixed crystals in the ball-milled mixture. Adding certain amounts of NaCl in MgCl{sub 2}.2.5EtOH adduct, either by co-milling or by simple mixing, greatly increased the thermal stability of the adduct, but thermal decomposition behaviour of the ball-milled mixture was still different from that of a simple mixture.

  2. Heating of food and haemoglobin adducts from carcinogens: possible precursor role of glycidol.

    PubMed

    Hindsø Landin, H; Tareke, E; Rydberg, P; Olsson, U; Törnqvist, M

    2000-11-01

    Studies of adducts from reactive compounds to haemoglobin (Hb) by gas chromatography-tandem mass spectrometry according to the N-alkyl Edman method reveals the occurrence of N-(2,3-dihydroxypropyl)valine (diHOPrVal) at levels of 1-2 pmol/g Hb, in persons without known exposure. The hypothesis that this background originates from glycidol or related compounds during heating of food was tested in experiments with rats. Animals fed fried animal feed for 30 or 72 days showed an increase of the diHOPrVal level by about 50% compared with controls. Several arguments, such as the formation of reactive oxiranes by heat-induced dehydration of glycol configurations in glycerol and sugars, support the idea that glycidol (or e.g. glycidyl esters) are precursors of the adduct. In Hb samples, reduced for stabilisation of aldehyde adducts, relatively high levels of adducts determined as diHOPrVal were found, although without significant relation to frying of the feed. There is thus no indication that reduction in vivo of, for example, the Schiff base from glyceraldehyde, is a pathway for formation of the diHOPrVal. The background level of diHOPrVal in humans Hb is low, and the cancer risk associated with exposure to the specific alkylator-probably glycidol-formed in cooking, is therefore presumably low. The result implies, however, that low-molecular mass mutagenic oxiranes formed during the heating of food should be studied further.

  3. Detection of Acetaminophen-Protein Adducts in Decedents with Suspected Opioid-Acetaminophen Combination Product Overdose.

    PubMed

    Thomas, Karen C; Wilkins, Diana G; Curry, Steven C; Grey, Todd C; Andrenyak, David M; McGill, Lawrence D; Rollins, Douglas E

    2016-09-01

    Acetaminophen overdose is a leading cause of drug-induced liver failure in the United States. Acetaminophen-protein adducts have been suggested as a biomarker of hepatotoxicity. The purpose of this study was to determine whether protein-derived acetaminophen-protein adducts are quantifiable in postmortem samples. Heart blood, femoral blood, and liver tissue were collected at autopsy from 22 decedents suspected of opioid-acetaminophen overdose. Samples were assayed for protein-derived acetaminophen-protein adducts, acetaminophen, and selected opioids found in combination products containing acetaminophen. Protein-derived APAP-CYS was detected in 17 of 22 decedents and was measurable in blood that was not degraded or hemolyzed. Heart blood concentrations ranged from 11 ng/mL (0.1 μM) to 7817 ng/mL (28.9 μM). Protein-derived acetaminophen-protein adducts were detectable in liver tissue for 20 of 22 decedents. Liver histology was also performed for all decedents, and no evidence of centrilobular hepatic necrosis was observed. PMID:27479586

  4. Poly(acrylic acid)/poly(ethylene glycol) adduct for attaining multifunctional cellulosic fabrics.

    PubMed

    Ibrahim, N A; Amr, A; Eid, B M; Mohamed, Z E; Fahmy, H M

    2012-06-20

    Aqueous polymerization of partially neutralized acrylic acid (AA) along with polyethylene glycol (PEG-600) at AA/PEG-600 mass ratio 3/1 using ammonium persulfate as initiator under proper conditions results in formation of PAA/PEG-600 adduct. The structure of the adduct was confirmed by FT-IR spectra. The potential applications of the prepared adduct in: sizing, durable hand building of cotton cellulose, as well as in functional finishing of cellulose containing fabrics, i.e. cotton, viscose and cotton/polyester, with Ag- or TiO2-nanoparticles were investigated. The modified substrates using the prepared adduct showed a remarkable improvement in their sizing, hand building and/or functional properties, i.e. antibacterial, anti-UV, and self cleaning, in addition to durability to wash. TEM images of the prepared nano-particles, SEM images of the untreated and treated substrates, as well as EDX spectra to analyze the surface elemental compositions were examined. The tentative mechanisms were also suggested. PMID:24750770

  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... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... 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, 2010 CFR

    2010-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  7. New adduct of abietane-type diterpene from Salvia leriifolia Benth.

    PubMed

    Hussain, Amjad; Adhikari, Achyut; Iqbal Choudhary, M; Ayatollahi, Syed Abdulmajid; Atta-Ur-Rahman

    2016-07-01

    A new adduct of abietane-type diterpene, salvialeriicone (1), was isolated from Salvia leriifolia Benth., along with a new chemical entity nor-abietane diterpene, 2-isopropyl-8,8-dimethyl-7,8-dihydrophenanthrene-1,4,5(6H)-trione (2). Their structures were determined using mass spectrometry, and 1D- and 2D-NMR spectroscopy.

  8. Separation and characterization of oxidized isomeric lipid-peptide adducts by ion mobility mass spectrometry.

    PubMed

    Milic, Ivana; Kipping, Marc; Hoffmann, Ralf; Fedorova, Maria

    2015-12-01

    Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic β1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. PMID:26634972

  9. Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

    PubMed

    Domingues, Rosário M; Domingues, Pedro; Melo, Tânia; Pérez-Sala, Dolores; Reis, Ana; Spickett, Corinne M

    2013-10-30

    Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. PMID:23770299

  10. Conformational, IR spectroscopic and electronic properties of conium alkaloids and their adducts with C60 fullerene

    NASA Astrophysics Data System (ADS)

    Zabolotnyi, M. A.; Prylutskyy, Yu I.; Poluyan, N. A.; Evstigneev, M. P.; Dovbeshko, G. I.

    2016-08-01

    Conformational, IR spectroscopic and electronic properties of the components of Conium alkaloids (Conium maculatum) in aqueous environment were determined by model calculations and experiment. With the help of FT-IR spectroscopy the possibility of formation of an adduct between γ-coniceine alkaloid and C60 fullerene was demonstrated, which is important for further application of conium analogues in biomedical purposes.

  11. 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. PMID:26650891

  12. Gender differences in hip adduction motion and torque during a single-leg agility maneuver.

    PubMed

    Hewett, Timothy E; Ford, Kevin R; Myer, Gregory D; Wanstrath, Kim; Scheper, Melia

    2006-03-01

    The purpose of this study was to identify gender differences in hip motion and kinetics during a single leg bidirectional deceleration maneuver. The rationale for the development of this maneuver was to test dynamic hip control during the deceleration of three different types of single-leg landings. The hypothesis was that female athletes would display increased hip adduction angles and moments during the maneuver compared to male athletes. Thirty-six collegiate soccer players (19 female, 17 male) volunteered to participate. Subjects were instructed to start the maneuver balancing on one foot, to hop through an agility-speed ladder on the same leg "up two boxes, back one, and then up one and hold it." Hip kinematics and kinetics during all three landings were examined. Females demonstrated significantly greater hip adduction angles at initial contact during all three landings and greater maximal hip adduction during landings 1 and 2 compared to male athletes. Females also exhibited significantly increased external hip adduction moments during landing 1, however, no differences were found between genders during landings 2 and 3.

  13. Inhibition of sulfotransferase affecting in vivo genotoxicity and DNA adducts induced by safrole in rat liver.

    PubMed

    Daimon, H; Sawada, S; Asakura, S; Sagami, F

    The effect of pretreatment with pentachlorophenol (PCP), a known inhibitor of sulfotransferases, on the induction of chromosomal aberrations, sister chromatid exchanges (SCEs), replicative DNA synthesis (RDS), and the formation of DNA adducts was studied in the liver of rats treated with safrole (1-allyl-3,4-methylenedioxy-benzene). Rats were given a single oral dose (1,000 mg/kg body weight) or 5 repeated doses (500 mg/kg body weight) of safrole, with or without intraperitoneal pretreatment with PCP (10 mg/kg body weight). Hepatocytes were isolated 24 hr after administration of safrole and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor to test for chromosomal aberrations and SCEs. For examination of RDS, hepatocytes were incubated in Williams' medium E containing 5-bromo-2'-deoxyuridine. Safrole-DNA adducts were detected by a nuclease P1-enhanced 32P-postlabeling assay. A single dose of safrole induced significant SCEs and RDS, while chromosomal aberrations were induced by 5 repeated doses. Two major and 2 minor DNA adducts were detected by both a single dose and 5 repeated doses. PCP significantly decreased safrole-induced cytogenetic effects and RDS, and caused a decrease in DNA adducts formed by safrole. These results suggest that safrole is capable of inducing SCEs, chromosomal aberrations, and RDS in the rat liver in vivo and that these effects may be induced by the sulfuric acid ester metabolite that can bind DNA.

  14. Noni juice reduces lipid peroxidation–derived DNA adducts in heavy smokers

    PubMed Central

    Wang, Mian-Ying; Peng, Lin; Jensen, Claude J; Deng, Shixin; West, Brett J

    2013-01-01

    Food plants provide important phytochemicals which help improve or maintain health through various biological activities, including antioxidant effects. Cigarette smoke–induced oxidative stress leads to the formation of lipid hydroperoxides (LOOHs) and their decomposition product malondialdehyde (MDA), both of which cause oxidative damage to DNA. Two hundred forty-five heavy cigarette smokers completed a randomized, double-blind, placebo-controlled clinical trial designed to investigate the effect of noni juice on LOOH- and MDA-DNA adducts in peripheral blood lymphocytes (PBLs). Volunteers drank noni juice or a fruit juice placebo every day for 1 month. DNA adducts were measured by 32P postlabeling analysis. Drinking 29.5–118 mL of noni juice significantly reduced adducts by 44.6–57.4%. The placebo, which was devoid of iridoid glycosides, did not significantly influence LOOH- and MDA-DNA adduct levels in current smokers. Noni juice was able to mitigate oxidative damage of DNA in current heavy smokers, an activity associated with the presence of iridoids. PMID:24804023

  15. MALDI-TOF analysis of steroid/PAH-modified DNA adducts at the femtomole level

    SciTech Connect

    Gooden, J.K.; Gross, M.L.; Stack, D.

    1995-12-31

    Covalent binding of polycyclic aromatic hydrocarbons (PAH`s) and steroids to DNA to form adducts is one of the first events in the process of tumor initiation in carcinogenesis. Structure elucidation and characterization of these adducts provide important information that leads to further understanding of their biological metabolic pathways. In in vivo and in vitro steroid/PAH-DNA binding studies, the reaction products (adducts) are often of low amount (low picomole to femtomole). Previous results from this laboratory have shown that the sensitivity of MALDI-TOF can be improved by proper matrix selection. An increase in sensitivity can also be obtained with the use of d-fucose as a co-matrix. In this study 4-phenyl-{alpha}-cyanocinnamic acid, PCC, 4-benzyloxy-{alpha}-cyanocinnamic acid, BCC, ferulic acid, FA, {alpha}-cyano-4-hydroxycinnamic acid, 4HCCA, and 3-(2-naphthyl)-2-cyanoacrylic NCA, were used in the determination of the limit of detection for two different DNA adducts dibenzocarbazole-5-N7Ade, and 4-hydroxyestrone-N7Gua.

  16. Mononuclear barium diketonate polyamine adducts. Synthesis, structures, and use in MOCVD of barium titanate

    SciTech Connect

    Gardiner, R.A.; Gordon, D.C.; Stauf, G.T.; Vaartstra, B.A.; Ostrander, R.L.; Rheingold, A.L.

    1994-11-01

    Mononuclear barium {beta}-diketonate Lewis base adducts have been synthesized by reaction of Ba(thd){sub 2} (thd = 2,2,6,6-tetramethyl-3,5-heptanedionate) with polyamines 1,1,4,7,7-pentamethyldiethylenetriamine (pmdt) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (hmtt). The adducts [Ba(thd){sub 2}(pmdt)] (I) and [Ba(thd){sub 2}(hmtt)] (II) have been characterized by NMR spectroscopy, elemental analyses and single-crystal X-ray diffraction. Compound I crystallizes in the space group P2{sub 1}/c with a = 10.577(3) {angstrom}, b = 23.547(7) {angstrom}, c = 15.963(5) {angstrom}, {beta} = 105.21(2){degrees}, and Z = 4. Compound II crystallizes in the space group P2{sub 1}/c with a = 10.833(6) {angstrom}, b = 20.442(12) {angstrom}, c = 19.404(9) {angstrom}, {beta} = 104.35(4){degrees}, and Z = 4. The adducts are seven- and eight-coordinate, respectively, with all nitrogen atoms of the polyamine bound to a single barium center. Compound I has been used for thin-film growth of BaTiO{sub 3} which has revealed that, compared to Ba(thd){sub 2}(tetraglyme), the polyamine adduct allows a larger temperature window for effective vapor transport. 10 refs., 2 figs., 2 tabs.

  17. The Utility of Naphthyl-Keratin Adducts as Biomarkers for Jet-Fuel Exposure

    EPA Science Inventory

    We investigated the association between biomarkers of dermal exposure, naphthyl-keratin adducts (NKA), and urine naphthalene biomarker levels in 105 workers routinely exposed to jet-fuel. A moderate correlation was observed between NKA and urine naphthalene levels (p = 0.061). Th...

  18. Creating Context for the Use of DNA Adduct Data in Risk Assessment

    EPA Science Inventory

    Assessments of human cancer risk require the integration of diverse types of data. Advancing technologies for quantitative measurements at the sub-cellular domain raise the critical issue of interpretation and use of DNA adduct data in context with current understanding of cancer...

  19. Determination of hemoglobin adducts in workers exposed to 2,4, 6-trinitrotoluene.

    PubMed

    Sabbioni, G; Wei, J; Liu, Y Y

    1996-07-12

    2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. TNT can be taken up through the skin and by inhalation. It is therefore essential to have fast and reliable methods to monitor human exposure. In rat experiments, it has been shown that TNT binds covalently to blood proteins and to tissue proteins. Hemoglobin (Hb) adducts of TNT are markers for the internal dose and possibly for the toxic effects of TNT, e.g. cataracts. In the present paper we introduce a new efficient method to quantify Hb adducts of TNT. Precipitated Hb was hydrolyzed with base in the presence of the surrogate internal standard 3,5-dinitroaniline (35DNA). The released 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT) were quantified against 35DNA by gas chromatography-mass spectrometry with negative-ion chemical ionization. Hb of 50 workers and controls from a Chinese munition factory were investigated. The Hb adduct levels ranged from 3.7 to 522 ng for 4ADNT and from 0 to 14.7 ng for 2ADNT per gram of Hb. However, in control samples from Germany no Hb adducts of 4ADNT or 2ADNT could be found.

  20. Haemoglobin adducts as biomarkers of occupational exposure to 1,3-butadiene.

    PubMed

    Osterman-Golkar, S; Peltonen, K; Anttinen-Klemetti, T; Landin, H H; Zorcec, V; Sorsa, M

    1996-03-01

    Adducts of 1,2-epoxy-3-butene (EB) with haemoglobin were monitored in 17 workers from the 1,3-butadiene (BD) production unit of a petrochemical plant and in nine referents employed at the same factory but not exposed to BD. The air concentrations of BD were determined using stationary and personal monitoring. The ambient level of exposure of the plant workers handling butadiene containers (sampling and voiding) was on average 11.2 +/- 18.6 (mean +/- SD) mg/m3. Maintenance and laboratory workers were exposed to levels < or = 1.2 mg/m3. The particular haemoglobin adduct measured was 2-hydroxy-3-butenylvaline, formed by reaction of N-terminal valine with carbon 1 in EB. The adduct levels were increased (0.16 +/- 0.099 pmol/g; n = 10) in plant workers compared with the levels in maintenance and laboratory workers and controls (approximately 0.05 pmol/g; seven laboratory workers and nine controls evaluated). Thus, the method used for adduct determination--derivatization of 200-300 mg globin samples with penta-fluorophenyl isothiocyanate according to the N-alkyl Edman method and detection of the thiohydantoin derivatives by tandem mass spectrometry--is sufficiently sensitive to allow monitoring of exposure to BD down to the p.p.m. level. PMID:8671730

  1. The use of lithiated adducts for structural analysis of acylglycerols by MS-ESI

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Electrospray ionization mass spectrometry (ESI-MS) using lithium adducts is the method of choice for the analysis of acyglycerols. The method can be used for the identification of the structures of fatty acid constituents, including the number and location of double bonds and hydroxyl groups. The me...

  2. DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles

    EPA Science Inventory

    AbstractContext: Biodiesel and biodiesel-blend fuels offer a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. Objectives: We compared the formation of covalent DNA adducts by the in vitro metabol...

  3. 40 CFR 721.465 - Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.465 Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The...

  4. 40 CFR 721.465 - Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.465 Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The...

  5. Lysine adducts between methyltetrahydrophthalic anhydride and collagen in guinea pig lung.

    PubMed

    Jönsson, B A; Wishnok, J S; Skipper, P L; Stillwell, W G; Tannenbaum, S R

    1995-11-01

    The formation of adducts between methyltetrahydrophthalic anhydride (MTHPA), an important industrial chemical and potent allergen, and collagen from guinea pig lung tissue was investigated. Collagen peptides were obtained from the lung tissue by homogenization, defatting, washing, and digestion with collagenase. In experiments in vitro, lung tissue was exposed to 8.4 mumol (50 microCi) of 14C MTHPA. The amount of adducts was 97 nmol MTHPA/g of wet tissue as determined from the bound radioactivity. In a study in vivo, four guinea pigs were injected intratracheally with 8.4 mumol of 14C MTHPA each. The amount of adducts was 0-1.2 nmol MTHPA/g of wet tissue (determined by bound radioactivity). N epsilon-methyltetrahydrophthaloyl-L-lysine (MTHPL) was synthesized and characterized by NMR, UV, and mass spectrometry (MS). A method to analyze MTHPL, after derivatization with methanol and pentafluorobenzoyl chloride, using gas chromatography-MS was developed. Analysis of Pronase-digested MTHPA-exposed lung tissue showed a concentration of 19 nmol MTHPL/g wet lung in vitro and between 0 and 0.15 nmol MTHPL/g wet lung in vivo. Thus, 20% in vitro and 12-15% in vivo of the bound radioactivity was found as adducts with lysine. These results are a first step toward studies of allergenic epitopes in proteins and methods for biological monitoring of exposure to acid anhydrides.

  6. Spin-trap-radical chromatography of spin adducts produced from L-valine by. gamma. -irradiation

    SciTech Connect

    Makiino, K.; Suzuki, N.; Moriya, F.; Rokushika, S.; Hatano, H.

    1980-01-01

    Diastereomeric spin adducts produced by reaction of 2-methyl-2-nitrosopropane with the short-lived radicals from L-valine by ..gamma..-irradiation could be separated and identified by means of high performance liquid chromatography and ESR spectroscopy. 6 figures.

  7. Plastids and Carotenoid Accumulation.

    PubMed

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    2016-01-01

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants. PMID:27485226

  8. Plastids and Carotenoid Accumulation.

    PubMed

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    2016-01-01

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants.

  9. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle.

    PubMed

    Kerwin, J L; Turecek, F; Xu, R; Kramer, K J; Hopkins, T L; Gatlin, C L; Yates, J R

    1999-03-15

    Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side

  10. Separation of {sup 32}P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC

    SciTech Connect

    King, L.C.; Gallagher, J.E.; Lewtas, J.; George, M.

    1994-07-01

    The {sup 32}P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO{sub 2}-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO{sub 2}-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO{sub 2}-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO{sub 2}-PAH HPLC gradient system was developed to separate NO{sub 2}-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO{sub 2}-PAH-DNA adducts were compared using both adduct enhancement versions of the {sup 32}P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO{sub 2}-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the {sup 32}P-postlabeled DNA adduct standards (PAHs and NO{sub 2}-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO{sub 2}-PAH standards used in this study. HPLC analyses of the NO{sub 2}-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.

  11. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    SciTech Connect

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

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves stacking of the AFB{sub 1

  12. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  13. Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin

    PubMed Central

    Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C.; Malle, Ernst; Sattler, Wolfgang

    2015-01-01

    Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl− system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood–brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120 min, decaying at a rate of 5.9 × 10−3 min−1. NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC–MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. PMID:25576489

  14. Quantitative analysis of positional isomers of triacylglycerols via electrospray ionization tandem mass spectrometry of sodiated adducts.

    PubMed

    Herrera, Lisandra Cubero; Potvin, Michael A; Melanson, Jeremy E

    2010-09-01

    Herein we report a reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC/MS/MS) method for the analysis of positional isomers of triacylglycerols (TAGs) in vegetable oils. The fragmentation behavior of [M + X](+) ions (X = NH(4), Li, Na or Ag) was studied on a quadrupole-time-of-flight (Q-TOF) mass spectrometer under low-energy collision-induced dissociation (CID) conditions. Mass spectra that were dependent on the X(+) ion and the nature and position of the acyl substituents were observed for four pairs of 'AAB/ABA'-type TAGs, namely PPO/POP, OOP/OPO, LLO/LOL and OOL/OLO (where P is 16:0, palmitic acid; O is 18:1, oleic acid; and L is 18:2, linoleic acid). For the majority of [M + X](+) adducts, the loss of the fatty acid in the outer positions (sn-1 or sn-3) was favored over the loss in the central position (sn-2), which enabled the determination of the fractional abundance of the isomers. Ratios of the intensity of fragment ions at various AAB/ABA compositions produced linear calibration curves with positive slopes, comparable to those obtained traditionally by ESI-MS/MS of [M + NH(4)](+) adducts. The only exceptions were the [M + Ag](+) adducts of the PPO/POP system, which produced calibration curves with negative slopes. Sodium adducts provided the most consistent level of isomeric discrimination for the TAGs studied and also offered the most convenience in that they required no additive to the mobile phase. Therefore, calibration curve data derived from [M + Na](+) adducts were applied to the quantification of TAG regioisomers in sunflower and olive oils. The regiospecific analysis showed that palmitic acid was typically located at positions sn-1 or sn-3, whereas unsaturated fatty acids, oleic and linoleic acids were mostly found at the sn-2 position. PMID:20814981

  15. In Vivo Kinematics of the Trapeziometacarpal Joint During Thumb Extension-flexion and Abduction-adduction

    PubMed Central

    Crisco, Joseph J.; Halilaj, Eni; Moore, Douglas C.; Patel, Tarpit; Weiss, Arnold-Peter C.; Ladd, Amy L.

    2014-01-01

    Purpose The primary aim of this study was to determine whether the in vivo kinematics of the trapeziometacarpal (TMC) joint differ as a function of age and sex during thumb extension-flexion and abduction-adduction motions. Methods The hands and wrists of 44 subjects (10 men and 11 women aged 18 to 35 years and 10 men and 13 women aged 40 to 75 years) with no symptoms or signs of TMC joint pathology were imaged with computed tomography (CT) during thumb extension, flexion, abduction, and adduction. The kinematics of the TMC joint were computed and compared across direction, age, and sex. Results We found no significant effects of age or sex, after normalizing for size, in any of the kinematic parameters. The extension-flexion and abduction-adduction rotation axes did not intersect, and both were oriented obliquely to the saddle-shaped anatomy of the TMC articulation. The extension-flexion axis was located in the trapezium and the abduction-adduction axis was located in the metacarpal. Metacarpal translation and internal rotation occurred primarily during extension-flexion. Discussion Our in vivo findings support previous cadaver and modeling studies that have concluded that the functional axes of the TMC joint are non-orthogonal and non-intersecting. However, in contrast to previous studies, we found extension-flexion and adduction-abduction to be coupled with internal-external rotation and translation. Specifically, internal rotation and ulnar translation were coupled with flexion, indicating a potential stabilizing screw-home mechanism. Clinical Relevance The treatment of TMC pathology and arthroplasty design require a detailed and accurate understanding of TMC function. This study confirms the complexity of TMC kinematics and describes metacarpal translation coupled with internal rotation during extension-flexion, which may explain some of the limitations of current treatment strategies and should help improve implant designs. PMID:25542440

  16. PAH-DNA adducts in cord blood and fetal and child development in a Chinese cohort

    SciTech Connect

    Tang, D.L.; Li, T.Y.; Liu, J.J.; Chen, Y.H.; Qu, L.R.; Perera, F.

    2006-08-15

    Polycyclic aromatic hydrocarbons (PAHs) are an important class of toxic pollutants released by fossil fuel combustion. Other pollutants include metals and particulate matter. PAH-DNA adducts, or benzo(a)pyrene (BaP) adducts as their proxy, provide a chemical-specific measure of individual biologically effective doses that have been associated with increased risk of cancer and adverse birth outcomes. In the present study we examined the relationship between prenatal PAH exposure and fetal and child growth and development in Tongliang, China, where a seasonally operated coal-fired power plant was the major pollution source. In a cohort of 150 nonsmoking women and their newborns enrolled between 4 March 2002 and 19 June 2002, BaP-DNA adducts were measured in maternal and umbilical cord blood obtained at delivery. High PAH-DNA adduct levels (above the median of detectable adduct level) were associated with decreased birth head circumference (p = 0.057) and reduced children's weight at 18 months, 24 months, and 30 months of age (p {lt} 0.05), after controlling for potential confounders. In addition, in separate models, longer duration of prenatal exposure was associated with reduced birth length (p = 0.033) and reduced children's height at 18 (p = 0.001), 24 (p {lt} 0.001), and 30 months of age (p {lt} 0.001). The findings suggest that exposure to elevated levels of PAHS, with the Tongliang power plant being a significant source, is associated with reduced fetal and child growth in this population.

  17. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

  18. 1,N(2)-propanodeoxyguanosine adduct formation in aortic DNA following inhalation of acrolein.

    PubMed Central

    Penn, A; Nath, R; Pan, J; Chen, L; Widmer, K; Henk, W; Chung, F L

    2001-01-01

    Recent reports indicate that many of the cytotoxic and health-threatening components of environmental tobacco smoke (ETS) reside in the vapor phase of the smoke. We have reported previously that inhalation of 1,3-butadiene, a prominent vapor phase component of ETS, accelerates arteriosclerotic plaque development in cockerels. In this study we asked whether inhaled acrolein, a reactive aldehyde that is also a prominent vapor-phase component of ETS, damages artery-wall DNA and accelerates plaque development. Cockerels inhaled 0, 1, or 10 ppm acrolein mixed with HEPA-filtered air for 6 hr. Half were killed immediately (day 1 group) for detection of the stable, premutagenic 1,N(2)-propanodeoxyguanosine acrolein adduct (AdG3) in aortic DNA via a (32)P-postlabeling/HPLC method, and half were killed after 10 days (day 10 group) for indirect assessment of adduct repair. In the day 1 group, acrolein-DNA adducts were 5 times higher in the 1 and 10 ppm groups than in HEPA-filtered air controls. However, in the day 10 group, adduct levels in the 1 and 10 ppm acrolein groups were reduced to the control adduct level. For the plaque studies, cockerels inhaled 1 ppm acrolein (6 hr/day, 8 weeks), mixed with the same HEPA-filtered air inhaled by controls. Plaque development was measured blind by computerized morphometry. Unlike butadiene inhalation, acrolein inhalation did not accelerate plaque development. Thus, even though repeated exposure to acrolein alone has no effect on plaque size under the exposure conditions described here, a single, brief inhalation exposure to acrolein elicits repairable DNA damage to the artery wall. These results suggest that frequent exposure to ETS may lead to persistent artery-wall DNA damage and thus provide sites on which other ETS plaque accelerants can act. PMID:11333181

  19. Role of CYP1B1 in PAH-DNA adduct formation and breast cancer risk

    SciTech Connect

    Goth-Goldstein, Regine; Russell, Marion L.; Muller, A.P.; Caleffi, M.; Eschiletti, J.; Graudenz, M.; Sohn, Michael D.

    2010-04-01

    This study investigated the hypothesis that increased exposure to polycyclic aromatic hydrocarbons (PAHs) increases breast cancer risk. PAHs are products of incomplete burning of organic matter and are present in cigarette smoke, ambient air, drinking water, and diet. PAHs require metabolic transformation to bind to DNA, causing DNA adducts, which can lead to mutations and are thought to be an important pre-cancer marker. In breast tissue, PAHs appear to be metabolized to their cancer-causing form primarily by the cytochrome P450 enzyme CYP1B1. Because the genotoxic impact of PAH depends on their metabolism, we hypothesized that high CYP1B1 enzyme levels result in increased formation of PAH-DNA adducts in breast tissue, leading to increased development of breast cancer. We have investigated molecular mechanisms of the relationship between PAH exposure, CYP1B1 expression and breast cancer risk in a clinic-based case-control study. We collected histologically normal breast tissue from 56 women (43 cases and 13 controls) undergoing breast surgery and analyzed these specimens for CYP1B1 genotype, PAH-DNA adducts and CYP1B1 gene expression. We did not detect any difference in aromatic DNA adduct levels of cases and controls, only between smokers and non-smokers. CYP1B1 transcript levels were slightly lower in controls than cases, but the difference was not statistically significant. We found no correlation between the levels of CYP1B1 expression and DNA adducts. If CYP1B1 has any role in breast cancer etiology it might be through its metabolism of estrogen rather than its metabolism of PAHs. However, due to the lack of statistical power these results should be interpreted with caution.

  20. In vivo genotoxicity and DNA adduct levels in the liver of rats treated with safrole.

    PubMed

    Daimon, H; Sawada, S; Asakura, S; Sagami, F

    1998-01-01

    The induction of chromosome aberrations, sister chromatid exchanges (SCEs), and the formation of DNA adducts was studied in hepatocytes of F344 rats exposed in vivo to safrole. Hepatocytes were isolated 24 h after a single dose of safrole or five repeated doses (once a day) by gastric intubation and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after 48 h in culture. Safrole-DNA adducts were detected by a nuclease P1-enhanced 32P-post-labeling assay in isolated hepatocytes from the rats. While a single dose was not sufficient to induce detectable levels of chromosome aberrations at the time of assay, five repeated doses induced these changes with a maximum frequency of 13.4%, compared with the control value of 1.8%. Both a single dose and five repeated doses induced significant SCEs, to a maximum frequency of 0.81 SCEs per chromosome, while the control value was 0.59 SCEs per chromosome. Two major and two minor DNA adducts were detected after treatment with either a single dose or five repeated doses. The maximum amount of total DNA adducts was 89.8 DNA adducts/10(7) nucleotides. These results show that safrole is a genotoxic carcinogen in the rat liver in vivo and suggest that the cytogenetic effects of this compound may result from covalent DNA modification in the rat liver. This in vivo cytogenetic assay should provide a useful means of evaluation of the genotoxicity of hepatocarcinogens.

  1. Time course of DNA adduct formation in peripheral blood granulocytes and lymphocytes after drinking alcohol

    PubMed Central

    Balbo, Silvia; Meng, Lei; Bliss, Robin L.; Jensen, Joni A.; Hatsukami, Dorothy K.; Hecht, Stephen S.

    2012-01-01

    Alcohol consumption is an established risk factor for cancers of the head and neck, colorectum, liver and female breast. Acetaldehyde, the primary metabolite of ethanol, is suspected to play a major role in alcohol-related carcinogenesis. Acetaldehyde binds to DNA resulting in formation of adducts. DNA adducts are involved in mutagenesis and carcinogenesis. N 2-Ethylidenedeoxyguanosine (N 2-ethylidene-dGuo) is the major adduct formed in this reaction. Studies have shown an association between alcohol drinking and levels of this DNA adduct, suggesting its potential use as a biomarker for studying alcohol-related carcinogenesis. However, there are no reports on the kinetics of formation and repair of N 2-ethylidene-dGuo after alcohol consumption. Therefore, we investigated levels of N 2-ethylidene-dGuo in DNA from human peripheral blood cells at several time points after consumption of increasing doses of alcohol. Ten healthy non-smokers were recruited and asked to abstain from alcohol consumption except for the study doses. The subjects were given measured doses of alcohol once a week for 3 weeks, targeting increasing blood alcohol levels. Blood was collected at several time points before and after each dose, DNA was isolated from granulocytes and lymphocytes and N 2-ethylidene-dGuo was quantified as its NaBH3CN reduction product N 2-ethyldeoxyguanosine by liquid chromatography–electrospray ionisation–tandem mass spectrometry. Significant increases in N 2-ethylidene-dGuo were observed after all doses and in both cell types. However, there was substantial intraindividual variability, indicating that there are other important sources of this adduct in peripheral blood DNA. Further studies are needed to better understand the origins of N 2-ethylidene-dGuo in blood cells, the exposures it reflects, and thus its potential use as a marker of alcohol’s genotoxic effects. PMID:22406526

  2. Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

    PubMed Central

    Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.

    2012-01-01

    Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account. PMID:22157354

  3. Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin.

    PubMed

    Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C; Malle, Ernst; Sattler, Wolfgang

    2015-02-15

    Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl(-) system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood-brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120min, decaying at a rate of 5.9×10(-3)min(-1). NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC-MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. PMID:25576489

  4. Chimpanzee accumulative stone throwing.

    PubMed

    Kühl, Hjalmar S; Kalan, Ammie K; Arandjelovic, Mimi; Aubert, Floris; D'Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M; Zuberbuehler, Klaus; Boesch, Christophe

    2016-01-01

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites.

  5. Chimpanzee accumulative stone throwing

    PubMed Central

    Kühl, Hjalmar S.; Kalan, Ammie K.; Arandjelovic, Mimi; Aubert, Floris; D’Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E.; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J.; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M.; Zuberbuehler, Klaus; Boesch, Christophe

    2016-01-01

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites. PMID:26923684

  6. Chimpanzee accumulative stone throwing.

    PubMed

    Kühl, Hjalmar S; Kalan, Ammie K; Arandjelovic, Mimi; Aubert, Floris; D'Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M; Zuberbuehler, Klaus; Boesch, Christophe

    2016-01-01

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites. PMID:26923684

  7. Measurement of glycidol hemoglobin adducts in humans who ingest edible oil containing small amounts of glycidol fatty acid esters.

    PubMed

    Honda, Hiroshi; Onishi, Masayuki; Fujii, Kenkichi; Ikeda, Naohiro; Yamaguchi, Tohru; Fujimori, Taketoshi; Nishiyama, Naohiro; Kasamatsu, Toshio

    2011-10-01

    Hemoglobin (Hb) adducts are frequently used to address and/or monitor exposure to reactive chemicals. Glycidol (G), a known animal carcinogen, has been reported to form Hb adducts. Here, we measure G adduct levels in humans who daily ingest DAG oil, an edible oil consisting mainly of diacylglycerol. Since DAG oil contains a small amount of glycidol fatty acid esters (GEs), possible exposure to G released from GEs has been raised as a possible concern. For measurement of Hb adducts, we employed the N-alkyl Edman method reported by Landin et al. (1996) using gas chromatography-tandem mass spectrometry with minor modifications to detect G-Hb adducts as N-(2,3-dihydroxy-propyl)valine (diHOPrVal). Blood samples were collected from 7 DAG oil users and 6 non-users, and then G-Hb adduct levels were measured. G-Hb adducts were detected in all samples. The average level of diHOPrVal was 3.5±1.9pmol/g globin in the DAG oil users and 7.1±3.1pmol/g globin in the non-users. We conclude that there is no increased exposure to G in individuals who daily ingest DAG oil.

  8. {sup 32}P-postlabeling analysis of DNA adducts in wild perch (Perca fluviatilis) and northern pike (Esox lucius)

    SciTech Connect

    Ericson, G.; Liewenborg, B.; Balk, L.

    1995-12-31

    Several previous studies have demonstrated a correlation between high concentrations of sediment-associated contaminants and elevated levels of aromatic/hydrophobic DNA adduct levels in the liver of benthic fish species. In the present study DNA adducts was analyzed in coastal populations of perch (Perca fluviatilis) and northern pike (Esox lucius). Fish were sampled from four different sites in a gradient from a heavily industrialized area at the Swedish Baltic coast. For comparison, fish were also caught in a reference area with no main industries and comparatively low levels of contaminants of anthropogenic origin. DNA was extracted from liver and several extrahepatic tissues and DNA adducts were analyzed by the nuclease PI version of the {sup 32}P-postlabeling assay. The autoradiograms derived from DNA of fish from the contaminated sites showed several adduct spots not visible on the autoradiograms derived from fish from the reference area. Total adduct levels were significantly elevated in several tissues in fish from contaminated sites compared to the reference area. Species and tissue-specific differences in adduct levels and the use of {sup 32}P-postlabeling analysis of DNA adducts as a biomarker to monitor the presence and effects of genotoxic chemicals in the aquatic environment are discussed.

  9. Screening for DNA Alkylation Mono and Cross-Linked Adducts with a Comprehensive LC-MS(3) Adductomic Approach.

    PubMed

    Stornetta, Alessia; Villalta, Peter W; Hecht, Stephen S; Sturla, Shana J; Balbo, Silvia

    2015-12-01

    A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology's neutral loss of the 2'-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS(2) fragmentation events and subsequent MS(3) fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS(2) fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by screening for DNA mono and cross-linked adducts in purified DNA and in DNA extracted from cells treated with PR104A, an experimental DNA alkylating nitrogen mustard prodrug currently under investigation for the treatment of leukemia. The results revealed the ability of this new DNA adductomic approach to detect anticipated and unknown PR104A-induced mono and cross-linked DNA adducts in biological samples. This methodology is expected to be a powerful tool for screening for DNA adducts induced by endogenous or exogenous exposures.

  10. Formation of DNA adducts in vitro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1-nitropyrene

    SciTech Connect

    Howard, P.C.; Heflich, R.H.; Evans, F.E.; Beland, F.A.

    1983-05-01

    The polycyclic nitroaromatic hydrocarbon 1-nitropyrene is an environmental pollutant, a potent bacterial mutagen, and a carcinogen. Xanthine oxidase, a mammalian nitroreductase, catalyzed the in vitro metabolic activation of this compound to DNA-bound adducts. Maximum adduct formation occurred at pH 5.5 to 6.0 and was increased by the addition of catalase to the incubation medium. DNA binding from 1-nitropyrene was inhibited by hydrogen peroxide, L-ascorbate, and glutathione. Enzymatic hydrolysis of the modified DNA and subsequent analysis by high-pressure liquid chromatography indicated the presence of one major and two minor adducts. The major adduct was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy as N-(deoxyguanosin-8-yl)-1-aminopyrene. The minor adducts appear to be decomposition products of the major adduct. When Salmonella typhimurium TA1538 was incubated with 1-nitropyrene, a strong correlation was found between the extent of DNA binding and the frequency of induced histidine reversions. Analysis of the bacterial DNA indicated one major adduct which had chromatographic properties and pKaS identical to those of N-(deoxyguanosin-8-yl)-1-aminopyrene. These data indicate that N-hydroxy-1-aminopyrene is probably the mutagenic and DNA-binding species formed during the metabolic reduction of 1-nitropyrene.

  11. Highly persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin: detection by 32P-postlabeling analysis.

    PubMed

    Randerath, E; Agrawal, H P; Reddy, M V; Randerath, K

    1983-08-01

    A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 mumol each of benzo[alpha]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 X 10(4) - 1.3 X 10(5) nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 X 10(6) - 2.7 X 10(6) nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins. PMID:6318965

  12. Charge separated states and singlet oxygen generation of mono and bis adducts of C60 and C70

    NASA Astrophysics Data System (ADS)

    Dallas, Panagiotis; Rogers, Gregory; Reid, Ben; Taylor, Robert A.; Shinohara, Hisanori; Briggs, G. Andrew D.; Porfyrakis, Kyriakos

    2016-02-01

    We present a series of fullerene derivatives and a study on their photoluminescence properties, complete with their efficiency as singlet oxygen generation photosensitizers. We demonstrate the intramolecular charge transfer between pyrene donor and fullerene acceptor. The opposite effect in decay lifetime measurements is observed for the mono and bis adducts of C60 and C70 for the first time, indicating an interplay between charge-separation and locally excited states. A monoexponential decay was observed for the mono adduct of C60 and the bis adduct of C70, while a biexponential decay was observed for the bis adduct of C60 and the mono adduct of C70. The effect of these molecules as sensitizers of the singlet oxygen radical was tested using detailed 3D excitation photoluminescence maps. A quenching of the singlet oxygen for the C60-mono and C70-bis adducts was observed while a strong photosensitizing effect was observed for the C60-bis and C70-mono adducts.

  13. The use of in vitro DNA adduct formation to estimate the genotoxicity of residues at contaminated sites.

    PubMed

    Shaw, G; Connell, D; Barron, W

    1995-05-01

    Genotoxic carcinogens such as polycyclic aromatic hydrocarbons (PAHs) covalently bind to the bases in DNA to form adducts. The formation of DNA adducts is significant with respect to chemical carcinogenesis. Many contaminated sites contain quantities of carcinogens such as PAHs, and the evaluation of the genotoxicity of these soils has important implications for human risk assessment. DNA adducts can be formed using an in vitro system incorporating extracts from contaminated soils. The 32P-postlabelling assay is a sensitive technique for the detection of DNA adducts from complex mixtures of environmental carcinogens. These techniques have been used to form and detect DNA adducts using soils from a number of coal gasworks sites. The results show that the extent of adduct formation depends partially on the petroleum hydrocarbon content of samples, but also on other undetermined factors related to composition. While environmental weathering has been shown to effect the PAH composition of samples, this is not an important factor in controlling the genotoxicity of samples as estimated by DNA adduct formation. PMID:7780722

  14. Adduct formation of 7,12-dimethylbenz(a)anthracene in the embryo of the Japanese medaka (Oryzias latipes)

    SciTech Connect

    Liu, H.; Cooper, K.R.

    1995-12-31

    DNA adduct formation of 7,1 2-dimethylbenz(a)anthracene (DMBA) in vivo in the Japanese medaka embryo were investigated using {sup 32}P-postlabeling analysis. 1-compounds (endogenous adducts) were not observed in the Japanese medaka embryo on days 4 (prior to liver formation), 6 (liver/swim bladder) or 10 (prior to hatch) of development. The level of DMBA:DNA adducts were concentration-dependent over the range of 0.625 ppm (Total Adducts 0.05707 pmol/mg of DNA) to 2.50 ppm (0.43341 pmol/mg of DNA) and decreased at 5.00 ppm (0.25338 pmol/mg of DNA) after medaka embryos were exposed to DMBA for 6 days from the day of fertilization. The decrease in DMBA:DNA adducts at 5.00 ppm was probably due to embryo toxicity (78% death). The level of DMBA:DNA adducts formed from the embryos exposed to DMBA for 24 hr decreased as the stage of development increased: day 4 > day 6 > day 10; 0.0262, 0.0179, 0.0129 pmol/mg of DNA, respectively. The level of DMBA:DNA adducts increased as the length of exposure increased: 4 day < 6 day < 10 day; 0.0233, 0.0614, 0.1502, respectively. There was both a time and dose dependence to the number of adducts detected. The data presented demonstrated the development of DM BA-DNA adducts in the developing Japanese medaka (Oryzias latipes) and the lack of I-compounds.

  15. Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.

    PubMed Central

    Napoli, C; D'Armiento, F P; Mancini, F P; Postiglione, A; Witztum, J L; Palumbo, G; Palinski, W

    1997-01-01

    To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs

  16. Modulatory effects of essential oils from spices on the formation of DNA adduct by aflatoxin B1 in vitro.

    PubMed

    Hashim, S; Aboobaker, V S; Madhubala, R; Bhattacharya, R K; Rao, A R

    1994-01-01

    Essential oils from common spices such as nutmeg, ginger, cardamom, celery, xanthoxylum, black pepper, cumin, and coriander were tested for their ability to suppress the formation of DNA adducts by aflatoxin B1 in vitro in a microsomal enzyme-mediated reaction. All oils were found to inhibit adduct formation very significantly and in a dose-dependent manner. The adduct formation appeared to be modulated through the action on microsomal enzymes, because an effective inhibition on the formation of activated metabolite was observed with each oil. The enzymatic modulation is perhaps due to the chemical constituents of the oils, and this could form a basis for their potential anticarcinogenic roles. PMID:8058527

  17. Accumulator with preclosing preventer

    SciTech Connect

    Murthy, R.R.; Rice, B.J.

    1981-11-24

    A guided-float accumulator suitable for use with a hydraulic system for an oil well blowout preventer is provided with a wing shut-off valve. Radially inwardly directed outlet parts are aimed at the bottom of the valve wing to generate unbalanced reaction forces which oppose the bernoulli effect forces caused by rapid movement of fluid through the chamber of the shut-off valve, thus preventing premature closing of the valve.

  18. Nuclear Magnetic Resonance Studies of an N2-Guanine Adduct Derived from the Tumorigen Dibenzo[a,l]pyrene in DNA: Impact of Adduct Stereochemistry, Size, and Local DNA Sequence on Solution Conformations

    PubMed Central

    2015-01-01

    The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide–DNA adducts formed include the stereoisomeric 14S and 14Rtrans-anti-DB[a,l]P-N2-dG and the stereochemically analogous 10S- and 10R-B[a]P-N2-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N2-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N2-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5′-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure–function relationship in NER. PMID

  19. Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensity

    PubMed Central

    Sharma, Purshotam; Manderville, Richard A.; Wetmore, Stacey D.

    2014-01-01

    The nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2′-deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the associated adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to determine the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helices were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic separation between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixture of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity and mutagenic outcomes, and support recent experiments suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochemical experiments on this potentially genotoxic lesion. PMID:25217592

  20. Site-specific synthesis of oligonucleotides containing malondialdehyde adducts of deoxyguanosine and deoxyadenosine via a postsynthetic modification strategy.

    PubMed

    Wang, Hao; Kozekov, Ivan D; Kozekova, Albena; Tamura, Pamela J; Marnett, Lawrence J; Harris, Thomas M; Rizzo, Carmelo J

    2006-11-01

    Malondialdehyde (MDA) and its reactive equivalent, base propenal, are products of oxidative damage to lipids and DNA, respectively; they are mutagenic in bacterial and mammalian systems, and MDA is carcinogenic in rats. MDA adducts of deoxyguanosine (M1dG), deoxyadenosine (OPdA), and deoxycytidine (OPdC) have been characterized. We have developed site-specific syntheses of M1dG and OPdA adducted oligonucleotides that rely on a postsynthetic modification strategy. This work provides an alternative route to the M1dG adducted oligonucleotide and, to date, the only viable strategy for the site-specific synthesis of OPdA-modified oligonucleotides. The stability of the modified oligonucleotides was examined by UV thermal melting studies (Tm). In contrast to the M1dG adduct, OPdA caused very little change in the Tm.

  1. Effects of bridge exercise on trunk core muscle activity with respect to sling height and hip joint abduction and adduction.

    PubMed

    Lee, Daehee; Park, Jungseo; Lee, Sangyong

    2015-06-01

    [Purpose] This study evaluated the effects of bridge exercise on trunk core muscle activity with respect to sling height and hip joint abduction and adduction. [Subjects] Fifteen healthy adult males participated. [Methods] In the bridge exercise, the height of the sling was set low or high during hip joint abduction and adduction. Electromyography was used to compare the differences between the muscle activities of the transverse abdominis, rectus abdominis, and erector spinae muscles. [Results] The muscle activities of the transverse abdominis, rectus abdominis, and erector spinae were significantly higher in the high sling position. Furthermore, the activities of the transverse abdominis and erector spinae were significantly higher during hip joint adduction than abduction regardless of sling height. [Conclusion] A high sling height is the most effective intervention for increasing the muscle activities of the transverse abdominis and erector spinae muscles during hip joint adduction in a bridge exercise. PMID:26180366

  2. Formation of an adduct between fosfomycin and glutathione: a new mechanism of antibiotic resistance in bacteria.

    PubMed Central

    Arca, P; Rico, M; Braña, A F; Villar, C J; Hardisson, C; Suárez, J E

    1988-01-01

    Plasmid-borne resistance to fosfomycin in bacteria is due to modification of the antibiotic molecule by a glutathione S-transferase that catalyzes the formation of a covalent bond between the sulfhydryl residue of the cysteine in glutathione and the C-1 of fosfomycin. This reaction results in opening of the epoxide ring of the antibiotic to form an inactive adduct, the structure of which was confirmed by nuclear magnetic resonance. Dialyzed extracts prepared from resistant Escherichia coli strains were unable to modify fosfomycin unless exogenous glutathione was added to the reaction mixtures. Similarly, mutants defective in glutathione biosynthesis were susceptible to fosfomycin, despite harboring a resistance plasmid. Extracts of resistant but not susceptible strains could join glutathione to 1-chloro-2,4-dinitrobenzene, confirming the nature of the enzymatic activity. Adduct formation appeared to be specific for glutathione: none of the other thiols tested (cysteine, N-acetylcysteine, and dithiothreitol) could modify fosfomycin. PMID:3056239

  3. 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.

  4. 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. PMID:26471529

  5. Lewis Acid-Base Adduct Approach for High Efficiency Perovskite Solar Cells.

    PubMed

    Lee, Jin-Wook; Kim, Hui-Seon; Park, Nam-Gyu

    2016-02-16

    Since the first report on the long-term durable 9.7% solid-state perovskite solar cell employing methylammonium lead iodide (CH3NH3PbI3), mesoporous TiO2, and 2,2',7,7'-tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9'-spirobifluorene (spiro-MeOTAD) in 2012, following the seed technologies on perovskite-sensitized liquid junction solar cells in 2009 and 2011, a surge of interest has been focused on perovskite solar cells due to superb photovoltaic performance and extremely facile fabrication processes. The power conversion efficiency (PCE) of perovskite solar cells reached 21% in a very short period of time. Such an unprecedentedly high photovoltaic performance is due to the intrinsic optoelectronic property of organolead iodide perovskite material. Moreover, a high dielectric constant, sub-millimeter scale carrier diffusion length, an underlying ferroelectric property, and ion migration behavior can make organolead halide perovskites suitable for multifunctionality. Thus, besides solar cell applications, perovskite material has recently been applied to a variety fields of materials science such as photodetectors, light emitting diodes, lasing, X-ray imaging, resistive memory, and water splitting. Regardless of application areas, the growth of a well-defined perovskite layer with high crystallinity is essential for effective utilization of its excellent physicochemical properties. Therefore, an effective methodology for preparation of high quality perovskite layers is required. In this Account, an effective methodology for production of high quality perovskite layers is described, which is the Lewis acid-base adduct approach. In the solution process to form the perovskite layer, the key chemicals of CH3NH3I (or HC(NH2)2I) and PbI2 are used by dissolving them in polar aprotic solvents. Since polar aprotic solvents bear oxygen, sulfur, or nitrogen, they can act as a Lewis base. In addition, the main group compound PbI2 is known to be a Lewis acid. Thus, PbI2 has a chance

  6. Synthesis of dithiafulvene-quinone donor-acceptor systems: isolation of a Michael adduct.

    PubMed

    Lissau, Henriette; Jevric, Martyn; Madsen, Anders Østergaard; Nielsen, Mogens Brøndsted

    2015-06-01

    π-Conjugated donor-acceptor systems based on dithiafulvene (DTF) donor units and various acceptor units have attracted attention for their linear and nonlinear optical properties. The reaction between p-benzoquinone and a 1,3-dithiole phosphonium salt, deprotonated by lithium hexamethyldisilazide (LiHMDS), gave a product mixture from which the Michael adduct [systematic name: dimethyl 2-(3-hydroxy-6-oxocyclohexa-2,4-dien-1-ylidene)-2H-1,3-dithiole-4,5-dicarboxylate], C13H10O6S2, was isolated. It is likely that one of the unidentified products obtained previously by others from related reactions could be a similar Michael adduct.

  7. Raman spectroscopic evaluation of DNA adducts of a platinum containing anticancer drug

    NASA Astrophysics Data System (ADS)

    Jangir, Deepak K.; Mehrotra, Ranjana

    2014-09-01

    Mechanistic understanding of the interaction of drugs with their target molecules is important for better understanding of their mode of action and to improve their efficacy. Carboplatin is a platinum containing anticancer drug, used to treat different type of tumors. In the present work, we applied Raman spectroscopy to study the interaction of carboplatin with DNA at molecular level using different carboplatin-DNA molar ratios. These Raman spectroscopic results provide comprehensive understanding on the carboplatin-DNA interactions and indicate that DNA cross-linked adducts formed by carboplatin are similar to cisplatin adducts. The results indicate that guanine N7 and adenine N7 are the putative sites for carboplatin interaction. It is observed that carboplatin has some affinity toward cytosine in DNA. Phosphate sugar backbone of DNA showed conformation perturbation in DNA which were easily sensible at higher concentrations of carboplatin. Most importantly, carboplatin interaction induces intermediate A- and B-DNA conformations at the cross-linking sites.

  8. Lewis Acid-Base Adduct Approach for High Efficiency Perovskite Solar Cells.

    PubMed

    Lee, Jin-Wook; Kim, Hui-Seon; Park, Nam-Gyu

    2016-02-16

    Since the first report on the long-term durable 9.7% solid-state perovskite solar cell employing methylammonium lead iodide (CH3NH3PbI3), mesoporous TiO2, and 2,2',7,7'-tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9'-spirobifluorene (spiro-MeOTAD) in 2012, following the seed technologies on perovskite-sensitized liquid junction solar cells in 2009 and 2011, a surge of interest has been focused on perovskite solar cells due to superb photovoltaic performance and extremely facile fabrication processes. The power conversion efficiency (PCE) of perovskite solar cells reached 21% in a very short period of time. Such an unprecedentedly high photovoltaic performance is due to the intrinsic optoelectronic property of organolead iodide perovskite material. Moreover, a high dielectric constant, sub-millimeter scale carrier diffusion length, an underlying ferroelectric property, and ion migration behavior can make organolead halide perovskites suitable for multifunctionality. Thus, besides solar cell applications, perovskite material has recently been applied to a variety fields of materials science such as photodetectors, light emitting diodes, lasing, X-ray imaging, resistive memory, and water splitting. Regardless of application areas, the growth of a well-defined perovskite layer with high crystallinity is essential for effective utilization of its excellent physicochemical properties. Therefore, an effective methodology for preparation of high quality perovskite layers is required. In this Account, an effective methodology for production of high quality perovskite layers is described, which is the Lewis acid-base adduct approach. In the solution process to form the perovskite layer, the key chemicals of CH3NH3I (or HC(NH2)2I) and PbI2 are used by dissolving them in polar aprotic solvents. Since polar aprotic solvents bear oxygen, sulfur, or nitrogen, they can act as a Lewis base. In addition, the main group compound PbI2 is known to be a Lewis acid. Thus, PbI2 has a chance

  9. Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation method

    SciTech Connect

    Toernqvist, M.M.; Mowrer, J.; Jensen, S.; Ehrenberg, L.

    1986-04-01

    Tissue doses of cancer initiators/mutagens are suitably monitored through hemoglobin adducts formed in vivo, but the use of this method has been hampered by a lack of sufficiently simple and fast procedures. It was previously observed that when the N-terminal amino acid in hemoglobin, valine, is alkylated it is cleaved off by the Edman sequencing reagent, phenyl isothiocyanate, in the neutral-alkaline coupling medium, as opposed to the acidic medium required by normal amino acids. Based on this principle, conditions for a functioning procedure for gas chromatography/mass spectrometry (GC/MS) determination of N-terminal alkylvalines in hemoglobin were worked out. Derivatizing the protein in formamide solution with pentafluorophenyl isothiocyanate, using a /sup 2/H-alkylated protein as internal standard, and applying on-column injection during analysis, permit reproducible determination of hydroxyethylvaline and other adducts down into the dose range where cancer risks may be considered acceptably low.

  10. Iridium-catalysed dehydrocoupling of aryl phosphine-borane adducts: synthesis and characterisation of high molecular weight poly(phosphinoboranes).

    PubMed

    Paul, Ursula S D; Braunschweig, Holger; Radius, Udo

    2016-06-30

    The thermal dehydrogenative coupling of aryl phosphine-borane adducts with iridium complexes bearing a bis(phosphinite) pincer ligand is reported. This catalysis produces high molecular weight poly(phosphinoboranes) [ArPH-BH2]n (Ar = Ph, (p)Tol, Mes). Furthermore, we investigated the reactivity of these pincer complexes towards primary phosphines and their respective borane adducts on a stoichiometric scale. PMID:27320239

  11. Ovarian susceptibility to benzo[a]pyrene: tissue burden of metabolites and DNA adducts in F-344 rats.

    PubMed

    Ramesh, Aramandla; Archibong, Anthony E; Niaz, Mohammad S

    2010-01-01

    Exposure to environmental toxicants has been implicated as one of the causative factors for infertility in mammals. The objective of this study was to determine the amount of ingested benzo[a]pyrene (BaP), an environmental toxicant that reaches the reproductive tissues (internal dose) subsequent to a single acute exposure. Toward this end, the concentrations of BaP reactive metabolites and BaP-DNA adducts were measured throughout the course of BaP's residence in the body. Ten-week-old female Fischer-344 rats weighing approximately 220 g were administered 5 mg BaP/kg body weight orally. 1, 7, 14, 2,1 and 28 d post BaP exposure, BaP parent compound and metabolites from plasma, ovaries, and liver tissues were extracted using liquid-liquid extraction. The extracts were analyzed by reverse-phase highperformance liquid chromatography (HPLC). DNA was isolated and analyzed for BaP-induced DNA adducts by (32)P-postlabeling method. The BaP total metabolite concentrations in plasma, ovaries, and liver showed a gradual decrease from d 1 to 28 post BaP administration. The BaP-DNA adducts concentrations in ovaries and liver tissues from the treatment group demonstrated a trend similar to that observed for metabolites. Ovaries showed greater concentrations of DNA adducts compared to liver. However, with an increase in time post cessation of exposure, the adduct concentrations in liver tissue started declining rapidly, from d 1 to 28. For ovaries, the adduct concentrations demonstrated a significant decline from d 1 to 7 and a gradual fall thereafter. A concordance between BaP reactive metabolite levels and adduct concentrations indicates that the bioavailability of reactive metabolites determines the binding with DNA and consequently the formation and persistence of adducts in an acute exposure regimen. PMID:20967675

  12. OVARIAN SUSCEPTIBILITY TO BENZO[a]PYRENE: TISSUE BURDEN OF METABOLITES AND DNA ADDUCTS IN F-344 RATS

    PubMed Central

    Ramesh, Aramandla; Archibong, Anthony E.; Niaz, Mohammad S.

    2010-01-01

    Exposure to environmental toxicants has been implicated as one of the causative factors for infertility in mammals. The objective of this study was to determine the amount of ingested benzo[a]pyrene (BaP), an environmental toxicant that reaches the reproductive tissues (internal dose) subsequent to a single acute exposure. Toward this end, the concentrations of BaP reactive metabolites and BaP–DNA adducts were measured throughout the course of BaP’s residence in the body. Ten-week-old female Fischer-344 rats weighing approximately 220 g were administered 5 mg BaP/kg body weight orally. 1, 7, 14, 2,1 and 28 d post BaP exposure, BaP parent compound and metabolites from plasma, ovaries, and liver tissues were extracted using liquid–liquid extraction. The extracts were analyzed by reverse-phase highperformance liquid chromatography (HPLC). DNA was isolated and analyzed for BaP-induced DNA adducts by 32P-postlabeling method. The BaP total metabolite concentrations in plasma, ovaries, and liver showed a gradual decrease from d 1 to 28 post BaP administration. The BaP–DNA adducts concentrations in ovaries and liver tissues from the treatment group demonstrated a trend similar to that observed for metabolites. Ovaries showed greater concentrations of DNA adducts compared to liver. However, with an increase in time post cessation of exposure, the adduct concentrations in liver tissue started declining rapidly, from d 1 to 28. For ovaries, the adduct concentrations demonstrated a significant decline from d 1 to 7 and a gradual fall thereafter. A concordance between BaP reactive metabolite levels and adduct concentrations indicates that the bioavailability of reactive metabolites determines the binding with DNA and consequently the formation and persistence of adducts in an acute exposure regimen. PMID:20967675

  13. Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose.

    PubMed

    Honda, Hiroshi; Törnqvist, Margareta; Nishiyama, Naohiro; Kasamatsu, Toshio

    2014-03-15

    Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo. Five groups of rats were orally administered a single dose of glycidol ranging from 0 to 75mg/kg bw, and diHOPrVal levels were measured 24h after administration. A dose-dependent increase in diHOPrVal levels was observed with high linearity (R(2)=0.943). Blood sampling at different time points (1, 10, 20, or 40days) from four groups administered glycidol at 12mg/kg bw suggested a linear decrease in diHOPrVal levels compatible with the normal turnover of rat erythrocytes (life span, 61days), with the calculated first-order elimination rate constant (kel) indicating that the diHOPrVal adduct was chemically stable. Then, we measured the second-order rate constant (kval) for the reaction of glycidol with N-terminal valine in rat and human hemoglobin in in vitro experiments with whole blood. The kval was 6.7±1.1 and 5.6±1.3 (pmol/g globin per μMh) in rat and human blood, respectively, indicating no species differences. In vivo doses estimated from kval and diHOPrVal levels were in agreement with the area under the (concentration-time) curve values determined in our earlier toxicokinetic study in rats. Our results indicate that diHOPrVal is a useful biomarker for quantification of glycidol exposure and for risk assessment.

  14. Free flow electrophoresis separation and AMS quantitation of C-naphthalene-protein adducts.

    PubMed

    Buchholz, Bruce A; Haack, Kurt W; Sporty, Jennifer L; Buckpitt, Alan R; Morin, Dexter

    2010-04-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of (14)C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with (14)C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts. PMID:20454606

  15. Aberrant methylation of hypermethylated-in-cancer-1 and exocyclic DNA adducts in tobacco smokers.

    PubMed

    Peluso, Marco E M; Munnia, Armelle; Bollati, Valentina; Srivatanakul, Petcharin; Jedpiyawongse, Adisorn; Sangrajrang, Suleeporn; Ceppi, Marcello; Giese, Roger W; Boffetta, Paolo; Baccarelli, Andrea A

    2014-01-01

    Tobacco smoke has been shown to produce both DNA damage and epigenetic alterations. However, the potential role of DNA damage in generating epigenetic changes is largely underinvestigated in human studies. We examined the effects of smoking on the levels of DNA methylation in genes for tumor protein p53, cyclin-dependent kinase inhibitor2A, hypermethylated-in-cancer-1 (HIC1), interleukin-6, Long Interspersed Nuclear Element type1, and Alu retrotransposons in blood of 177 residents in Thailand using bisulfite-PCR andpyrosequencing. Then, we analyzed the relationship of this methylation with the oxidative DNA adduct, M₁dG (a malondialdehyde adduct), measured by ³²P-postlabeling. Multivariate statistical analyses showed that HIC1 methylation levels were significantly increased in smokers compared with nonsmokers (p ≤ .05). A dose response was observed, with the highest HIC1 methylation levels in smokers of ≥ 10 cigarettes/day relative to nonsmokers and intermediate values in smokers of 1-9 cigarettes/day (p for trend ≤ .001). No additional relationships were observed. We also evaluated correlations between M₁dG and the methylation changes at each HIC1 CpG site individually. The levels of this adduct in smokers showed a significant linear correlation with methylation at one of the 3 CpGs evaluated in HIC1: hypermethylation at position 1904864340 was significantly correlated with the adduct M₁dG (covariate-adjusted regression coefficient (β) = .224 ± .101 [SE], p ≤ .05). No other correlations were detected. Our study extends prior work by others associating hypermethylation of HIC1 with smoking; shows that a very specific hypermethylation event can arise from smoking; and encourages future studies that explore a possible role for M₁dG in connecting smoking to this latter hypermethylation. PMID:24154486

  16. Analytical determination of specific 4,4'-methylene diphenyl diisocyanate hemoglobin adducts in human blood.

    PubMed

    Gries, Wolfgang; Leng, Gabriele

    2013-09-01

    4,4'-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4'-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography-high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population. PMID:23839327

  17. Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts

    PubMed Central

    Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter

    2009-01-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5–11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts. PMID:20454606

  18. Electrochemical oxidation and protein adduct formation of aniline: a liquid chromatography/mass spectrometry study.

    PubMed

    Melles, Daniel; Vielhaber, Torsten; Baumann, Anne; Zazzeroni, Raniero; Karst, Uwe

    2012-04-01

    Historically, skin sensitization tests are typically based on in vivo animal tests. However, for substances used in cosmetic products, these tests have to be replaced according to the European Commission regulation no. 1223/2009. Modification of skin proteins by electrophilic chemicals is a key process associated with the induction of skin sensitization. The present study investigates the capabilities of a purely instrumental setup to determine the potential of commonly used non-electrophilic chemicals to cause skin sensitization by the generation of electrophilic species from the parent compound. In this work, the electrophiles were generated by the electrochemical oxidation of aniline, a basic industrial chemical which may also be released from azo dyes in cosmetics. The compound is a known sensitizer and was oxidized in an electrochemical thin-layer cell which was coupled online to electrospray ionization-mass spectrometry. The electrochemical oxidation was performed on a boron-doped diamond working electrode, which is able to generate hydroxyl radicals in aqueous solutions at high potentials. Without any pretreatment, the oxidation products were identified by electrospray ionization/time-of-flight mass spectrometry (ESI-ToF-MS) using their exact masses. A mass voltammogram was generated by plotting the obtained mass spectra against the applied potential. Oligomerization states with up to six monomeric units in different redox states of aniline were observed using this setup. This approach was extended to generate adducts between the oxidation products of aniline and the tripeptide glutathione. Two adducts were identified with this trapping experiment. Protein modification was carried out subsequently: Aniline was oxidized at a constant potential and was allowed to react with β-lactoglobulin A (β-LGA) or human serum albumin (HSA), respectively. The generated adducts were analyzed by liquid chromatography coupled to ESI-ToF-MS. For both β-LGA and HSA, aniline

  19. Correlation between the knee adduction torque and medial contact force for a variety of gait patterns.

    PubMed

    Zhao, Dong; Banks, Scott A; Mitchell, Kim H; D'Lima, Darryl D; Colwell, Clifford W; Fregly, Benjamin J

    2007-06-01

    The external knee adduction torque has been proposed as a surrogate measure for medial compartment load during gait. However, a direct link between these two quantities has not been demonstrated using in vivo measurement of medial compartment load. This study uses in vivo data collected from a single subject with an instrumented knee implant to evaluate this link. The subject performed five different overground gait motions (normal, fast, slow, wide, and toe-out) with simultaneous collection of instrumented implant, video motion, and ground reaction data. For each trial, the knee adduction torque was measured externally while the total axial force applied to the tibial insert was measured internally. Based on data collected from the same subject performing treadmill gait under fluoroscopic motion analysis, a regression equation was developed to calculate medial contact force from the implant load cell measurements. Correlation analyses were performed for the stance phase and entire gait cycle to quantify the relationship between the knee adduction torque and both the medial contact force and the medial to total contact force ratio. When the entire gait cycle was analyzed, R(2) for medial contact force was 0.77 when all gait trials were analyzed together and between 0.69 and 0.93 when each gait trial was analyzed separately (p < 0.001 in all cases). For medial to total force ratio, R(2) was 0.69 for all trials together and between 0.54 and 0.90 for each trial separately (p < 0.001 in all cases). When only the stance phase was analyzed, R(2) values were slightly lower. These results support the hypothesis that the knee adduction torque is highly correlated with medial compartment contact force and medial to total force ratio during gait.

  20. Assessment of long-term health risks after accidental exposure using haemoglobin adducts of epichlorohydrin.

    PubMed

    Wollin, Klaus-Michael; Bader, Michael; Müller, Michael; Lilienblum, Werner; Csicsaky, Michael

    2014-12-15

    On September 9th, 2002, two goods trains collided in Bad Münder, Lower Saxony, causing the release of more than 40 metric tonnes of epichlorohydrin (1-chloro-2,3-epoxypropane) into the environment. A human biomonitoring study was performed to evaluate the accidental exposure to epichlorohydrin and to assess the possible long-term, i.e. carcinogenic health effects. This was done on the basis of a biochemical effect monitoring using the N-(3-chloro-2-hydroxypropyl)valine and the N-(2,3-dihydroxypropyl)valine haemoglobin adducts of epichlorohydrin in blood to respond to missing ambient monitoring immediately after the crash. N-(3-chloro-2-hydroxypropyl)valine adduct levels above the LOQ (25 pmol/g globin) ranged from 32.0 to 116.4 pmol/g globin in 6 out of 628 samples. The N-(2,3-dihydroxypropyl)valine adduct was not detected above the LOD (10 pmol/g globin) in any of the blood samples. Based on the quantified N-(3-chloro-2-hydroxypropyl)valine adduct values, the body doses after two days of exposure were estimated to be in the range of 1.7-6.2 nmol/kg body weight. The reverse estimation of the external exposure leads to cumulative additional lifetime cancer risks ranging from 2.61×10(-8) to 9.48×10(-8). The estimated excess lifetime cancer risks have to be assessed as extremely low. Our biomonitoring study facilitated the dialogue between individuals and groups concerned and authorities, because suspected or occurred exposures and risks to human health could be quantified and interpreted in a sound manner.

  1. 2[prime] and 3[prime] Carboranyl uridines and their diethyl ether adducts

    DOEpatents

    Soloway, A.H.; Barth, R.F.; Anisuzzaman, A.K.; Alam, F.; Tjarks, W.

    1992-12-15

    A process is described 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. The carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of the compounds in methods for boron neutron capture therapy in mammalian tumor cells. No Drawings

  2. Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts

    NASA Astrophysics Data System (ADS)

    Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter

    2010-04-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

  3. Charge transfer states in stable neutral and oxidized radical adducts from carbazole derivatives.

    PubMed

    Fajarí, Lluís; Papoular, Robert; Reig, Marta; Brillas, Enric; Jorda, José Luis; Vallcorba, Oriol; Rius, Jordi; Velasco, Dolores; Juliá, Luis

    2014-02-21

    In this paper we report the spectral properties of the stable radical adducts 1(•)-3(•), which are formed by an electron donor moiety, the carbazole ring, and an electron acceptor moiety, the polychlorotriphenylmethyl radical. The molecular structure of radical adduct 1(•) in the crystalline state shows a torsion angle of approximately 90° between the phenyl and the carbazole rings due to steric interactions. They exhibit a charge transfer band in the visible range of the electronic spectrum. All of them are chemically oxidized with copper(II) perchlorate to the respective cation species, which show a strong charge transfer band into the near-infrared region of the spectrum. Radical adducts 1(•)-3(•) and the corresponding stable oxidized species 1(+)-3(+) are real organic mixed-valence compounds due to the open-shell nature of their electronic structure. Charge transfer bands of the cation species are stronger and are bathochromically shifted with respect to those of the neutral species due to the greater acceptor ability of the positively charged central carbon atom of the triphenylmethyl moiety. The cationic species 1(+)-3(+) are diamagnetic, as shown by the absence of a signal in the EPR spectrum in acetonitrile solution at room temperature, but they show an intense and unique band in frozen solutions (183 K).

  4. Molecular basis of aflatoxin-induced mutagenesis—role of the aflatoxin B1-formamidopyrimidine adduct

    PubMed Central

    Lin, Ying-Chih; Li, Liang; Makarova, Alena V.; Burgers, Peter M.; Stone, Michael P.; Lloyd, R. Stephen

    2014-01-01

    Aflatoxin B1 (AFB1) is a known carcinogen associated with early-onset hepatocellular carcinoma (HCC) and is thought to contribute to over half a million new HCCs per year. Although some of the fundamental risk factors are established, the molecular basis of AFB1-induced mutagenesis in primate cells has not been rigorously investigated. To gain insights into genome instability that is produced as a result of replicating DNAs containing AFB1 adducts, site-specific mutagenesis assays were used to establish the mutagenic potential of the persistent ring-opened AFB1 adduct, AFB1-formamidopyrimidine (AFB1-FAPY). This lesion was highly mutagenic, yielding replication error frequencies of 97%, with the predominant base substitution being a G to T transversion. This transversion is consistent with previous mutational data derived from aflatoxin-associated HCCs. In vitro translesion synthesis assays demonstrated that polymerase (pol) ζ was the most likely candidate polymerase that is responsible for the G to T mutations induced by this adduct. PMID:24398669

  5. Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

    PubMed Central

    Yee, Estella F.; Diensthuber, Ralph P.; Vaidya, Anand T.; Borbat, Peter P.; Engelhard, Christopher; Freed, Jack H.; Bittl, Robert; Möglich, Andreas; Crane, Brian R.

    2015-01-01

    Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications. PMID:26648256

  6. Chemical discrimination between dC and 5MedC via their hydroxylamine adducts.

    PubMed

    Münzel, Martin; Lercher, Lukas; Müller, Markus; Carell, Thomas

    2010-11-01

    The presence of the methylated nucleobase (5Me)dC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of (5Me)dC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and (5Me)dC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the (5Me)dC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and (5Me)dC with high accuracy, leading towards a novel and mild chemistry for methylation analysis.

  7. Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue.

    PubMed

    Yee, Estella F; Diensthuber, Ralph P; Vaidya, Anand T; Borbat, Peter P; Engelhard, Christopher; Freed, Jack H; Bittl, Robert; Möglich, Andreas; Crane, Brian R

    2015-12-09

    Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

  8. Supramolecular Adducts of Cucurbit[7]uril and Amino Acids in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Kovalenko, Ekaterina; Vilaseca, Marta; Díaz-Lobo, Mireia; Masliy, A. N.; Vicent, Cristian; Fedin, Vladimir P.

    2016-02-01

    The complexation of the macrocyclic cavitand cucurbit[7]uril (Q7) with a series of amino acids (AA) with different side chains (Asp, Asn, Gln, Ser, Ala, Val, and Ile) is investigated by ESI-MS techniques. The 1:1 [Q7 + AA + 2H]2+ adducts are observed as the base peak when equimolar Q7:AA solutions are electrosprayed, whereas the 1:2 [Q7 + 2AA + 2H]2+ dications are dominant when an excess of the amino acid is used. A combination of ion mobility mass spectrometry (IM-MS) and DFT calculations of the 1:1 [Q7 + AA + 2H]2+ (AA = Tyr, Val, and Ser) adducts is also reported and proven to be unsuccessful at discriminating between exclusion or inclusion-type conformations in the gas phase. Collision induced dissociation (CID) revealed that the preferred dissociation pathways of the 1:1 [Q7 + AA + 2H]2+ dications are strongly influenced by the identity of the amino acid side chain, whereas ion molecule reactions towards N-butylmethylamine displayed a common reactivity pattern comprising AA displacement. Special emphasis is given on the differences between the gas-phase behavior of the supramolecular adducts with amino acids (AA = Asp, Asn, Gln, Ser, Ala, Val, and Ile) and those featuring basic (Lys and Arg) and aromatic (Tyr and Phe) side chains.

  9. Toxicological evaluation of acyl glucuronides utilizing half-lives, peptide adducts, and immunostimulation assays.

    PubMed

    Iwamura, Atsushi; Ito, Masahito; Mitsui, Hideaki; Hasegawa, Jun; Kosaka, Keigo; Kino, Ichiro; Tsuda, Minoru; Nakajima, Miki; Yokoi, Tsuyoshi; Kume, Toshiyuki

    2015-12-25

    Chemical reactivity of acyl glucuronides (AGs) is believed to be involved in the toxicity of carboxylic acid-containing drugs. Both direct and immune-mediated toxicity have been suggested as possible mechanisms of toxicity; however, it remains unclear. In the present study, we performed assays of half-lives, peptide adducts, and immunostimulation to evaluate the potential risk of AGs of 21 drugs and analyzed the relationship to the toxic category. AGs of all withdrawn drugs tested in this study showed short half-lives and peptide adducts formation, but so did those of several safe drugs. In contrast, only AGs of withdrawn and warning drugs induced interleukin-8 (IL-8) in human peripheral blood mononuclear cells (hPBMCs). Using a DNA microarray assay, we found that zomepirac AG induced the mRNAs of 5 genes, including IL-8 in hPBMCs. In addition, withdrawn and warning drugs were distinguished from safe drugs by an integrated score of relative mRNA expression levels of 5 genes. The immunostimulation assay showed higher sensitivity, specificity, and accuracy compared with other methods. In preclinical drug development, the evaluation of the reactivity of AGs using half-lives and peptide adducts assays followed by the evaluation of immunostimulation by highly reactive AGs using hPBMCs can contribute to improved drug safety.

  10. Theoretical study of coupling p-aminothiophenol to hydroazo- and azo-adducts on Au(111).

    PubMed

    Lang, Xiufeng; Liang, Yanhong; Liu, Siyan; Zhao, Shanshan; Lau, Woon-Ming

    2016-09-01

    Aminothiophenol/Au(111) has been adopted as an exemplary model in plasmonics research, including surface-enhanced Raman spectroscopy, due to its high plasmonic-induced spectral-signal enhancement. The present work was aimed at clarifying whether aminothiophenol on Au(111) is chemically stable in the absence of any photo- and plasmonic-induced effects. Briefly, first-principles calculations were employed to track the detailed mechanism of oxidative coupling of p-aminothiophenol (PATP) to its azo-adduct with an N = N bond, i.e., p,p'-dimercaptoazobenzene (DMAB). Our results show the following: first, in the presence of adsorbed O2, PATP fractures its N-H bond and transfers the hydrogen to a nearby oxygen. This pathway is more favorable than the transfer of H to Au, but the activation barrier of 0.9 eV is still too high for the reaction to occur in the absence of thermal-, photo-, or plasmonic-activation. If this bar can be lifted, two such dehydrogenated PATP can couple themselves to form an adduct with a N-N bond, i.e., p,p'-dimercaptohydroazobenzene (DMHAB), and this reaction is exoergic with an energy barrier of 0.57 eV. Again, this step is slow in the absence of moderate thermal activation or photo-/plasmonic-activation. Finally, dehydrogenation of DMHAB gives the azo-adduct of DMAB, and this reaction is spontaneous, with no energy barrier. PMID:27488103

  11. Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

    PubMed Central

    Czerwińska, Jolanta; Poznański, Jarosław; Dębski, Janusz; Bukowy, Zuzanna; Bohr, Vilhelm A.; Tudek, Barbara; Speina, Elżbieta

    2014-01-01

    4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence. PMID:25170083

  12. Process for making a calcium/sodium ferrate adduct by the electrochemical formation of sodium ferrate

    SciTech Connect

    Deininger, J.P.; Dotson, R.L.

    1984-05-29

    Described is a process for making a calcium/sodium ferrate adduct with sodium ferrate in a divided-type electrolysis cell. The anolyte chamber of the cell is charged with an aqueous solution of sodium hydroxide and a sodium ferrate-stabilizing proportion of at least one sodium halide salt. The anolyte chamber additionally contains ferric ions (Fe(III)). The catholyte chamber contains an aqueous sodium hydroxide solution during operation. The source of ferric ion in the anolyte may be either an iron-containing anode or at least one iron-containing compound present in the anolyte solution or both. The preferred material separating the anolyte chamber from the catholyte chamber is comprised of a gas- and hydraulic-impermeable, ionically-conductive, chemically-stable ionomeric film (e.g., a cation-exchange membrane with carboxylic, sulfonic or other inorganic exchange sites). Sodium ferrate is prepared in the anolyte chamber by passing an electric current and impressing a voltage between the anode and cathode of the cell. During electrolysis, sodium ferrate forms in the aqueous sodium hydroxide anolyte. This anolyte is reacted with a calcium compound to produce a calcium/sodium ferrate adduct. Alternatively the sodium ferrate may be first recovered in a solid form and then reacted with a calcium compound to produce said adduct.

  13. Characterization and Reactivity of a Terminal Nickel(III)-Oxygen Adduct

    DOE PAGES

    Pirovano, Paolo; Farquhar, Erik R.; Swart, Marcel; Fitzpatrick, Anthony J.; Morgan, Grace G.; McDonald, Aidan R.

    2015-01-22

    Here, high-valent terminal metal–oxygen adducts are hypothesized to be the potent oxidizing reactants in late transition metal oxidation catalysis. In particular, examples of high-valent terminal nickel–oxygen adducts are scarce, meaning there is a dearth in the understanding of such oxidants. A monoanionic NiII-bicarbonate complex has been found to react in a 1:1 ratio with the one-electron oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, yielding a thermally unstable intermediate in high yield (ca. 95%). Electronic absorption, electronic paramagnetic resonance, and X-ray absorption spectroscopies and density functional theory calculations confirm its description as a low-spin (S=1/2), square planar NiIII–oxygen adduct. Moreover, this rare example of amore » high-valent terminal nickel–oxygen complex performs oxidations of organic substrates, including 2,6-di-tert-butylphenol and triphenylphosphine, which are indicative of hydrogen atom abstraction and oxygen atom transfer reactivity, respectively.« less

  14. Characterization and Reactivity of a Terminal Nickel(III)-Oxygen Adduct

    SciTech Connect

    Pirovano, Paolo; Farquhar, Erik R.; Swart, Marcel; Fitzpatrick, Anthony J.; Morgan, Grace G.; McDonald, Aidan R.

    2015-01-22

    Here, high-valent terminal metal–oxygen adducts are hypothesized to be the potent oxidizing reactants in late transition metal oxidation catalysis. In particular, examples of high-valent terminal nickel–oxygen adducts are scarce, meaning there is a dearth in the understanding of such oxidants. A monoanionic NiII-bicarbonate complex has been found to react in a 1:1 ratio with the one-electron oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, yielding a thermally unstable intermediate in high yield (ca. 95%). Electronic absorption, electronic paramagnetic resonance, and X-ray absorption spectroscopies and density functional theory calculations confirm its description as a low-spin (S=1/2), square planar NiIII–oxygen adduct. Moreover, this rare example of a high-valent terminal nickel–oxygen complex performs oxidations of organic substrates, including 2,6-di-tert-butylphenol and triphenylphosphine, which are indicative of hydrogen atom abstraction and oxygen atom transfer reactivity, respectively.

  15. The effect of vary varus malalignment on knee adduction moment during walking of human normal gait.

    PubMed

    Maneekittichot, T; Sorachaimetha, P; Onmanee, P; Chanthasopeephan, T

    2013-01-01

    This research aims to study the effect of varus malalignment to knee adduction moment (KAM) during walking using 3D gait simulation. KAM is the product of frontal ground reaction force and frontal lever arm; it is a major cause of pain at the lateral knee that is the general symptom of osteoarthritis (OA). For treatment, lateral fixed wedge insole and variable-stiffness shoes were used to treat OA patient for many years. The device helps reduce KAM while walking by shifting the center of pressure (CoP) from medial side to lateral side. Therefore, shifting CoP to lateral side for reducing frontal lever arm was incorporated into the design of the treatment devices for OA patient. In this paper, program simulation "Adams life module" was used to create 3D human model and simulate 3D gait to observe changes of KAM while vary the adduction angle between thigh and tibia. The simulation model was created based on normal gait profile data during the movement of the model. The result obtained from the simulation showed that the varus malalignment plays important roles on KAM. Increasing of the adduction angle leads to the higher value of peak KAM during walking.

  16. Carbofuran poisoning detected by mass spectrometry of butyrylcholinesterase adduct in human serum.

    PubMed

    Li, He; Ricordel, Ivan; Tong, Larry; Schopfer, Lawrence M; Baud, Frédéric; Mégarbane, Bruno; Maury, Eric; Masson, Patrick; Lockridge, Oksana

    2009-03-01

    Carbofuran is a pesticide whose acute toxicity is due to inhibition of acetylcholinesterase. Butyrylcholinesterase (BChE) in plasma is inhibited by carbofuran and serves as a biomarker of poisoning by carbofuran. The goal was to develop a method to positively identify poisoning by carbofuran. Sera from an attempted murder and an attempted suicide were analyzed for the presence of carbofuran adducts on BChE. The BChE from 1 ml of serum was rapidly purified on a 0.2 ml procainamide-Sepharose column. Speed was essential because the carbofuran-BChE adduct decarbamylates with a half-life of about 2 h. The partially purified BChE was boiled to denature the protein, thus stopping decarbamylation and making the protein vulnerable to digestion with trypsin. The labeled peptide was partially purified by HPLC before analysis by LC/MS/MS in the multiple reaction monitoring mode on the QTRAP 2000 mass spectrometer. Carbofuran was found to be covalently bound to Ser 198 of human BChE in serum samples from two poisoning cases. Multiple reaction monitoring triggered MS/MS spectra positively identified the carbofuran-BChE adduct. In conclusion a mass spectrometry method to identify carbofuran poisoning in humans has been developed. The method uses 1 ml of serum and detects low-level exposure associated with as little as 20% inhibition of plasma butyrylcholinesterase.

  17. A three-dimensional model of vocal fold abduction/adduction

    NASA Astrophysics Data System (ADS)

    Hunter, Eric J.; Titze, Ingo R.; Alipour, Fariborz

    2004-04-01

    A three-dimensional biomechanical model of tissue deformation was developed to simulate dynamic vocal fold abduction and adduction. The model was made of 1721 nearly incompressible finite elements. The cricoarytenoid joint was modeled as a rocking-sliding motion, similar to two concentric cylinders. The vocal ligament and the thyroarytenoid muscle's fiber characteristics were implemented as a fiber-gel composite made of an isotropic ground substance imbedded with fibers. These fibers had contractile and/or passive nonlinear stress-strain characteristics. The verification of the model was made by comparing the range and speed of motion to published vocal fold kinematic data. The model simulated abduction to a maximum glottal angle of about 31°. Using the posterior-cricoarytenoid muscle, the model produced an angular abduction speed of 405° per second. The system mechanics seemed to favor abduction over adduction in both peak speed and response time, even when all intrinsic muscle properties were kept identical. The model also verified the notion that the vocalis and muscularis portions of the thyroarytenoid muscle play significantly different roles in posturing, with the muscularis portion having the larger effect on arytenoid movement. Other insights into the mechanisms of abduction/adduction were given.

  18. Protein adducts of the prostate carcinogen PhIP in children

    SciTech Connect

    Lawrence Livermore National Laboratory

    2004-02-20

    Prostate cancer is the second leading cause of cancer death in men in the United States. few epidemiology studies have indicated that exposure to PhIP, a rodent prostate carcinogen formed in meat during cooking, may be an important risk factor for prostate cancer in humans. Therefore, a highly sensitive biomarker assay is urgently needed to clarify the role of PhIP in prostate cancer. The goal of this project is to develop an assay that can be used to more accurately quantify human exposure to PhIP and potential prostate cancer risk. Our hypothesis is that an Accelerator Mass Spectrometry-based method can be developed to measure protein adducts of PhIP in the blood of humans. This will provide a measure of the internal dose, as well as the capacity for carcinogen bioactivation to a form that can initiate the cancer process. Towards this goal, we have characterized an adduct formed by PhIP in vitro with the amino acid cysteine. This adduct should provide a biomarker of dietary PhIP exposure and potential prostate cancer risk that could be used to identify individuals for prevention and for monitoring the effect chemoprevention strategies.

  19. Benzo(a)pyrene diolepoxide-DNA adducts detected by synchronous fluorescence spectrophotometry.

    PubMed Central

    Vahakangas, K; Trivers, G; Rowe, M; Harris, C C

    1985-01-01

    Using benzo(a)pyrene (BP) as a model carcinogen we are currently applying a fluorescence technique to detect the very low levels of carcinogen-DNA adducts in human populations due to environmental exposure. In synchronous fluorescence spectrophotometry for detection of BP-diol epoxide-DNA, excitation and emission wavelengths are scanned simultaneously with a fixed wavelength difference (delta lambda) of 34 nm. Compared to conventional fluorescence methods only one peak emerges because excitation and emission peaks have to match delta lambda to show. Because of the quenching effect of DNA, samples are hydrolyzed by acid. After this, BP-diol epoxide (BPDE)- -modified DNA gives a peak at the same wavelength and of the same fluorescence yield as BP-tetrols. When DNA from peripheral blood lymphocytes of 44 coke oven workers were analyzed, 10 had a sharp peak at 379. Among 36 coke oven workers from another factory, 4 had detectable levels of adducts. A much smaller percentage of samples was positive in a group of aluminum plant workers. We have also found BPDE-DNA adducts in DNA from pulmonary alveolar macrophages and peripheral blood lymphocytes from tobacco smokers and some of the nonsmokers. PMID:3936704

  20. Molecular structures of five adducts assembled from p-dimethylaminobenzaldehyde and organic acids

    NASA Astrophysics Data System (ADS)

    Jin, Shouwen; Wang, Lanqing; Liu, Hui; Liu, Li; Zhang, Huan; Wang, Daqi; Li, Minghui; Guo, Jianzhong; Guo, Ming

    2016-07-01

    Five adducts 1-5 derived from p-dimethylaminobenzaldehyde have been prepared and characterized by X-ray diffraction analysis, IR, mp, and elemental analysis. Of the five adducts two are organic salts (1, and 2) and the other three (3-5) are cocrystals. In salts 1, and 2, the L molecules are protonated. The supramolecular architectures of the adducts 1-5 involve extensive intermolecular N-H⋯O, O-H⋯O, O-H⋯S, and C-H⋯O hydrogen bonds as well as other non-covalent interactions. The role of weak and strong non-covalent interactions in the crystal packing is ascertained. The complexes displayed 2D/3D framework structure for the synergistic effect of the various non-covalent interactions. The results presented herein tell that the strength and directionality of the N-H⋯O, O-H⋯O, and O-H⋯S hydrogen bonds between organic acids and p-dimethylaminobenzaldehyde are sufficient to bring about the formation of binary cocrystals or organic salts.

  1. Chemical discrimination between dC and 5MedC via their hydroxylamine adducts

    PubMed Central

    Münzel, Martin; Lercher, Lukas; Müller, Markus; Carell, Thomas

    2010-01-01

    The presence of the methylated nucleobase 5MedC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of 5MedC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and 5MedC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the 5MedC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and 5MedC with high accuracy, leading towards a novel and mild chemistry for methylation analysis. PMID:20813757

  2. Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma.

    PubMed

    Johannesson, Gunvor A; Kristiansson, Monica H; Jönsson, Bo A G; Lindh, Christian H

    2008-03-01

    An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.

  3. Tumors and DNA adducts in mice exposed to benzo[a]pyrene and coal tars: implications for risk assessment.

    PubMed Central

    Goldstein, L S; Weyand, E H; Safe, S; Steinberg, M; Culp, S J; Gaylor, D W; Beland, F A; Rodriguez, L V

    1998-01-01

    Current methods to estimate the quantitative cancer risk of complex mixtures of polycyclic aromatic hydrocarbons (PAH) such as coal tar assume that overall potency can be derived from knowledge of the concentration of a few carcinogenic components such as benzo[a]pyrene (B[a]P). Genotoxic damage, such as DNA adducts, is thought to be an essential aspect of PAH-induced tumorigenesis and could be a biomarker for exposure useful for estimating risk. However, the role of B[a]P and the relationship of adduct formation in tumorigenesis have not been tested rigorously in models appropriate for human health risk assessment. Therefore, we directly compared tumor induction and adduct formation by B[a]P and coal tars in several experimental protocols, including one broadly accepted and used by regulators. We found that B[a]P content did not account for tumor incidences after exposure to coal tars. DNA adducts were found in both tumors and tumor-free tissue and tumor outcomes were not predicted by either quantitation of total DNA adducts or by the DNA adduct formed by B[a]P. These data suggest that risk assessments based on B[a]P content may not predict accurately risk to human health posed by environmental PAH. PMID:9860888

  4. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore.

    PubMed

    Perera, Rukshan T; Fleming, Aaron M; Johnson, Robert P; Burrows, Cynthia J; White, Henry S

    2015-02-20

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5' or 3' directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  5. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

    NASA Astrophysics Data System (ADS)

    Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

    2015-02-01

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  6. Effect of 2, 5-Substituents on the Stability of Cyclic Nitrone Superoxide Spin Adducts: A Density Functional Theory Approach

    PubMed Central

    Du, Li-Bo; Wang, Lan-Fen; Liu, Yang-Ping; Jia, Hong-Ying; Liu, Yang; Liu, Ke Jian; Tian, Qiu

    2011-01-01

    To design efficient spin traps for superoxide radicals, interest in the elucidation of substituent effects on the stability of superoxide spin adducts has become a necessary priority. In the present study, five cyclic nitrone superoxide spin adducts, i.e. DMPO-OOH, M3PO-OOH, EMPO-OOH, DEPMPO-OOH, and DEPDMPO-OOH, were chosen as model compounds to investigate the effect of 2,5-subsitituents on their stability, through structural analysis and decay thermodynamics using density functional theory (DFT) calculations. Analysis of the optimized geometries reveals that none of the previously proposed stabilizing factors, including intramolecular H-bonds, intramolecular nonbonding interactions, bulky steric protection, nor the C(2)–N(1) bond distance can be used to clearly explain the effect of 2,5-substituents on the stability of the spin adducts. Additionally the effect of the 2,5-substituents on the stability of the superoxide spin adducts cannot be simply clarified by Milliken charges on both atoms (nitroxyl nitrogen and nitroxyl oxygen). Subsequent study found that spin densities on the nitroxyl nitrogen and oxygen are well correlated with the half-life times of the spin adducts, and consequently are the proper parameters to characterize the effect of 2,5-substituents on their stability. Examination of the decomposition thermodynamics further supports the effect of the substituents on the persistence of cyclic nitrone superoxide spin adducts. PMID:20370568

  7. Extraction of rare earth oxides using supercritical carbon dioxide modified with Tri-n-butyl phosphate–nitric acid adducts

    DOE PAGES

    Baek, Donna L.; Fox, Robert V.; Case, Mary E.; Sinclair, Laura K.; Schmidt, Alex B.; McIlwain, Patrick R.; Mincher, Bruce J.; Wai, Chien M.

    2016-06-14

    A new tri-n-butylphosphate–nitric acid (TBP–HNO3) adduct was prepared by combining TBP and fuming (90%) HNO3. The adduct was characterized, and its phase-equilibrium behavior in supercritical carbon dioxide is reported. Supercritical carbon dioxide (sc-CO2) was modified with this new adduct [TBP(HNO3)5.2(H2O)1.7], and the extraction efficacies of selected rare earth oxides (Y, Ce, Eu, Tb, and Dy) at 338 K and 34.5 MPa were compared with those obtained using an adduct formed from concentrated (70%) HNO3 and TBP [TBP(HNO3)1.7(H2O)0.6]. All rare earth oxides tested with both adduct species could be extracted with the exception of cerium oxide. Furthermore, the water and acidmore » concentrations in the different adducts were found to play a significant role in rare earth oxide extraction efficiency.« less

  8. The formation of lipid hydroperoxide-derived amide-type lysine adducts on proteins: a review of current knowledge.

    PubMed

    Kato, Yoji

    2014-01-01

    Lipid peroxidation is an important biological reaction. In particular, polyunsaturated fatty acid (PUFA) can be oxidized easily. Peroxidized lipids often react with other amines accompanied by the formation of various covalent adducts. Novel amide-type lipid-lysine adducts have been identified from an in vitro reaction mixture of lipid hydroperoxide with a protein, biological tissues exposed to conditions of oxidative stress and human urine from a healthy person. In this chapter, the current knowledge of amide type adducts is reviewed with a focus on the evaluation of functional foods and diseases with a history of discovery of hexanoyl-lysine (HEL). Although there is extensive research on HEL and other amide-type adducts, the mechanism of generation of the amide bond remains unclear. We have found that the decomposed aldehyde plus peroxide combined with a lysine moiety does not fully explain the formation of the amide-type lipid-lysine adduct that is generated by lipid hydroperoxide. Singlet oxygen or an excited state of the ketone generated from the lipid hydroperoxide may also contribute to the formation of the amide linkage. The amide-adducts may prove useful not only for the detection of oxidative stress induced by disease but also for the estimation of damage caused by an excess intake of PUFA. PMID:24374915

  9. Molecular characterization of the boron adducts of the proteasome inhibitor Bortezomib with epigallocatechin-3-gallate and related polyphenols

    PubMed Central

    Glynn, Stephen J.; Gaffney, Kevin J.; Sainz, Marcos A.; Louie, Stan G.

    2015-01-01

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) was reported to effectively antagonize the ability of Bortezomib to induce apoptosis in cancer cells. This interaction was attributed to the formation of a covalent adduct between a phenolic moiety of EGCG with the boronic acid group of Bortezomib. However, the structural details of this boron adduct and the molecular factors that contribute to its formation and its ability to inhibit Bortezomib's activity remain unclear. This paper describes the use of NMR spectroscopy and cell assays to characterize the structures and properties of the boron adducts of EGCG and related polyphenols. The observed boron adducts included both boronate and borate derivatives, and their structural characteristics were correlated with cell-based evaluation of the ability of EGCG and other phenols to antagonize the anticancer activity of Bortezomib. The enhanced stability of the BZM/EGCG adduct was attributed to electronic and steric reasons, and a newly identified intramolecular interaction of the boron atom of BZM with the adjacent amide bond. The reported approach provides a useful method for determining the potential ability of polyphenols to form undesired adducts with boron-based drugs and interfere with their actions. PMID:25669488

  10. DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells

    PubMed Central

    Attaluri, Sivaprasad; Bonala, Radha R.; Yang, In-Young; Lukin, Mark A.; Wen, Yujing; Grollman, Arthur P.; Moriya, Masaaki; Iden, Charles R.; Johnson, Francis

    2010-01-01

    Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ∼50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA→T and AL-II-dG→T transversions. PMID:19854934

  11. Heat exchanger-accumulator

    DOEpatents

    Ecker, Amir L.

    1980-01-01

    What is disclosed is a heat exchanger-accumulator for vaporizing a refrigerant or the like, characterized by an upright pressure vessel having a top, bottom and side walls; an inlet conduit eccentrically and sealingly penetrating through the top; a tubular overflow chamber disposed within the vessel and sealingly connected with the bottom so as to define an annular outer volumetric chamber for receiving refrigerant; a heat transfer coil disposed in the outer volumetric chamber for vaporizing the liquid refrigerant that accumulates there; the heat transfer coil defining a passageway for circulating an externally supplied heat exchange fluid; transferring heat efficiently from the fluid; and freely allowing vaporized refrigerant to escape upwardly from the liquid refrigerant; and a refrigerant discharge conduit penetrating sealingly through the top and traversing substantially the length of the pressurized vessel downwardly and upwardly such that its inlet is near the top of the pressurized vessel so as to provide a means for transporting refrigerant vapor from the vessel. The refrigerant discharge conduit has metering orifices, or passageways, penetrating laterally through its walls near the bottom, communicating respectively interiorly and exteriorly of the overflow chamber for controllably carrying small amounts of liquid refrigerant and oil to the effluent stream of refrigerant gas.

  12. LC-MS/MS screening strategy for unknown adducts to N-terminal valine in hemoglobin applied to smokers and nonsmokers.

    PubMed

    Carlsson, Henrik; von Stedingk, Hans; Nilsson, Ulrika; Törnqvist, Margareta

    2014-12-15

    Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC-MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503-638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.

  13. Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose

    SciTech Connect

    Honda, Hiroshi; Törnqvist, Margareta; Nishiyama, Naohiro; Kasamatsu, Toshio

    2014-03-15

    Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo. Five groups of rats were orally administered a single dose of glycidol ranging from 0 to 75 mg/kg bw, and diHOPrVal levels were measured 24 h after administration. A dose-dependent increase in diHOPrVal levels was observed with high linearity (R{sup 2} = 0.943). Blood sampling at different time points (1, 10, 20, or 40 days) from four groups administered glycidol at 12 mg/kg bw suggested a linear decrease in diHOPrVal levels compatible with the normal turnover of rat erythrocytes (life span, 61 days), with the calculated first-order elimination rate constant (k{sub el}) indicating that the diHOPrVal adduct was chemically stable. Then, we measured the second-order rate constant (k{sub val}) for the reaction of glycidol with N-terminal valine in rat and human hemoglobin in in vitro experiments with whole blood. The k{sub val} was 6.7 ± 1.1 and 5.6 ± 1.3 (pmol/g globin per μMh) in rat and human blood, respectively, indicating no species differences. In vivo doses estimated from k{sub val} and diHOPrVal levels were in agreement with the area under the (concentration–time) curve values determined in our earlier toxicokinetic study in rats. Our results indicate that diHOPrVal is a useful biomarker for quantification of glycidol exposure and for risk assessment. - Highlight: • Glycidol-hemoglobin adduct (diHOPrVal) was characterized for exposure evaluation. • We studied the kinetics of diHOPrVal formation and elimination in vitro and in vivo. • Dose dependent formation and chemical stability were confirmed in the rat study. • In vivo dose (AUC) of glycidol could be estimated from diHOPrVal levels

  14. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  15. Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark.

    PubMed

    von Stedingk, Hans; Vikström, Anna C; Rydberg, Per; Pedersen, Marie; Nielsen, Jeanette K S; Segerbäck, Dan; Knudsen, Lisbeth E; Törnqvist, Margareta

    2011-11-21

    The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography-mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/maternal hemoglobin adduct level ratios were 0.48 (range 0.27-0.86) for acrylamide, 0.38 (range 0.20-0.73) for glycidamide, and 0.43 (range 0.17-1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38-0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large

  16. Relationships among Polycyclic Aromatic Hydrocarbon–DNA Adducts, Proximity to the World Trade Center, and Effects on Fetal Growth

    PubMed Central

    Perera, Frederica P.; Tang, Deliang; Rauh, Virginia; Lester, Kristin; Tsai, Wei Yann; Tu, Yi Hsuan; Weiss, Lisa; Hoepner, Lori; King, Jeffrey; Del Priore, Giuseppe; Lederman, Sally Ann

    2005-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants released by the World Trade Center (WTC) fires and various urban combustion sources. Benzo[a]pyrene (BaP) is a representative member of the class of PAHs. PAH–DNA adducts, or BaP–DNA adducts as their proxy, provide a measure of chemical-specific genetic damage that has been associated with increased risk of adverse birth outcomes and cancer. To learn whether PAHs from the WTC disaster increased levels of genetic damage in pregnant women and their newborns, we analyzed BaP–DNA adducts in maternal (n = 170) and umbilical cord blood (n = 203) obtained at delivery from nonsmoking women who were pregnant on 11 September 2001 and were enrolled at delivery at three downtown Manhattan hospitals. The mean adduct levels in cord and maternal blood were highest among newborns and mothers who resided within 1 mi of the WTC site during the month after 11 September, intermediate among those who worked but did not live within this area, and lowest in those who neither worked nor lived within 1 mi (reference group). Among newborns of mothers living within 1 mi of the WTC site during this period, levels of cord blood adducts were inversely correlated with linear distance from the WTC site (p = 0.02). To learn whether PAHs from the WTC disaster may have affected birth outcomes, we analyzed the relationship between these outcomes and DNA adducts in umbilical cord blood, excluding preterm births to reduce variability. There were no independent fetal growth effects of either PAH–DNA adducts or environmental tobacco smoke (ETS), but adducts in combination with in utero exposure to ETS were associated with decreased fetal growth. Specifically, a doubling of adducts among ETS-exposed subjects corresponded to an estimated average 276-g (8%) reduction in birth weight (p = 0.03) and a 1.3-cm (3%) reduction in head circumference (p = 0.04). The findings suggest that exposure to elevated levels of PAHs, indicated by PAH

  17. Solids Accumulation Scouting Studies

    SciTech Connect

    Duignan, M. R.; Steeper, T. J.; Steimke, J. L.

    2012-09-26

    The objective of Solids Accumulation activities was to perform scaled testing to understand the behavior of remaining solids in a Double Shell Tank (DST), specifically AW-105, at Hanford during multiple fill, mix, and transfer operations. It is important to know if fissionable materials can concentrate when waste is transferred from staging tanks prior to feeding waste treatment plants. Specifically, there is a concern that large, dense particles containing plutonium could accumulate in poorly mixed regions of a blend tank heel for tanks that employ mixing jet pumps. At the request of the DOE Hanford Tank Operations Contractor, Washington River Protection Solutions, the Engineering Development Laboratory of the Savannah River National Laboratory performed a scouting study in a 1/22-scale model of a waste staging tank to investigate this concern and to develop measurement techniques that could be applied in a more extensive study at a larger scale. Simulated waste tank solids: Gibbsite, Zirconia, Sand, and Stainless Steel, with stainless steel particles representing the heavier particles, e.g., plutonium, and supernatant were charged to the test tank and rotating liquid jets were used to mix most of the solids while the simulant was pumped out. Subsequently, the volume and shape of the mounds of residual solids and the spatial concentration profiles for the surrogate for heavier particles were measured. Several techniques were developed and equipment designed to accomplish the measurements needed and they included: 1. Magnetic particle separator to remove simulant stainless steel solids. A device was designed and built to capture these solids, which represent the heavier solids during a waste transfer from a staging tank. 2. Photographic equipment to determine the volume of the solids mounds. The mounds were photographed as they were exposed at different tank waste levels to develop a composite of topographical areas. 3. Laser rangefinders to determine the volume of

  18. Ozonation of DNA forms adducts: a 32P-DNA labeling and thin-layer chromatography technique to measure DNA environmental biomarkers.

    PubMed

    Cajigas, A; Gayer, M; Beam, C; Steinberg, J J

    1994-01-01

    Little direct documented evidence of ozone's genotoxicity exists. Deoxyribonucleic acid (DNA) adducts are produced by environmental toxic agents, including ozone. We have described a modified thin-layer chromatography (TLC) technique that can assess adduct formation as a biomarker of ozone injury. This requires 32P-labeling DNA, digestion of deoxynucleotides (dNMPs), and separation in two-dimensional PEI-cellulose TLC. We have applied this technique to control DNAs, to control DNA in solution exposed to acute ambient ozone, and to control DNA exposed to acute bubbled-through ozone (2 ppm for 24 h). We detected stable DNA adducts, including hydroxymethyluracil (HMU), thymine glycol (TG), 8-hydroxyguanine (8-OHG), and demonstrated, as yet, unidentified adducts that may serve as a "fingerprint" pattern of DNA adduction. This technique quantifies low-molecular-mass DNA adducts, both in vivo and in vitro, with potential applications to environmental toxicology.

  19. Accumulation of radioactivity after repeated infusion of 3H-adrenaline and 3H-noradrenaline in the rat as a model animal.

    PubMed

    Lepschy, M; Filip, T; Palme, R G

    2014-10-01

    Besides enzymatic inactivation, catecholamines bind non-enzymatically and irreversible to proteins. The physiological impact of these catecholamine adducts is still unclear. We therefore collected basic data about the distribution of catecholamine adducts in the rat after repeated intravenous administration of (3)H-adrenaline and (3)H-noradrenaline. In all animals radioactivity in blood increased until the last injection on Day 7 and decreased then slowly close to background values (plasma) or remained higher (erythrocytes). In all sampled tissues radioactivity could be found, but only in hair high amounts remained present even after 3 weeks. Half-life of rat serum albumin loaded with (3)H-adrenaline or (3)H-noradrenaline was not altered. This study provides basic knowledge about the distribution of catecholamines or their adducts, but physiological effects could not be demonstrated. However, for the first time deposition and accumulation of catecholamines (adducts) in the hair could be proven, suggesting that hair might be used for evaluating long term stress.

  20. 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

  1. Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion.

    PubMed

    Tsuge, Kouichiro; Seto, Yasuo

    2006-06-21

    To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis. PMID:16569519

  2. Hemoglobin adducts of the human bladder carcinogen o-toluidine after treatment with the local anesthetic prilocaine.

    PubMed

    Gaber, Kerstin; Harréus, Ulrich A; Matthias, Christoph; Kleinsasser, Norbert H; Richter, Elmar

    2007-01-01

    Prilocaine, a widely used local anesthetic, is metabolized to o-toluidine which is classified as human carcinogen. We aimed to assess the impact of prilocaine-treatment on hemoglobin adducts from o-toluidine. Blood samples were obtained before and 24h after receiving prilocaine local anesthesia (Xylonest, 100mg) from 20 head and neck surgery patients and 6 healthy volunteers. Hemoglobin adducts of o-toluidine and 4-aminobiphenyl were determined by gas chromatography/mass spectrometry. Hemoglobin adducts of o-toluidine were significantly increased 24h after 100mg prilocaine-treatment by 21.6+/-12.8ng/g hemoglobin (mean+/-S.D., N=26; P<0.0001). This corresponds to a 6-360-fold increase of o-toluidine adduct levels in 25 patients from 0.54+/-0.95ng/g before treatment to 22.0+/-13.2ng/g 24h after surgery (mean+/-S.D.). Because of an extremely high background level the increase was only 1.6-fold in one patient (40.9ng/g before and 64.4ng/g 24h after prilocaine injection). Current smoking had no influence on background values and on the increase of o-toluidine adducts. No treatment-related differences were seen in mean hemoglobin adduct levels of 4-aminobiphenyl which were significantly higher in smokers, 0.149+/-0.096ng/g (mean+/-S.D., N=8) as compared to nonsmokers 0.036+/-0.035ng/g (mean+/-S.D., N=16; P<0.01). In conclusion, prilocaine anesthesia leads to a massive increase of hemoglobin adducts of the carcinogenic arylamine o-toluidine. This implies a carcinogenic risk which should be taken into account in preventive hazard minimization.

  3. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    SciTech Connect

    Ganesan, Shanthi Keating, Aileen F.

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  4. Biomonitoring of smoke constituents: exposure to 4-aminobiphenyl and 4-aminobiphenyl hemoglobin adduct levels in nonsmokers and smokers.

    PubMed

    Schorp, Matthias K; Leyden, Donald E

    2010-08-01

    Public health authorities worldwide have concluded that exposure to environmental tobacco smoke (ETS) causes diseases, including cancer, in adult nonsmokers. The arylamine, 4-aminobiphenyl (4-ABP), has been identified as a human carcinogen. Some publications have suggested that 4-ABP hemoglobin (4-ABP-Hb) adduct levels in nonsmokers are a result of exposure to ETS, whereas others could not confirm these observations. Toxicokinetic and exposure models proposed in this work are used to estimate the concentration of 4-ABP-Hb adducts resulting from ETS exposure that is based on experimental values for respirable suspended particulates (RSP) concentration. Monte Carlo methods were used to obtain estimates of population distributions of 4-ABP-Hb adduct levels resulting from indoor ETS exposure in homes, workplaces, and hospitality environments. It is found that the mean, median, and 95th percentile 4-ABP-Hb adduct steady-state levels of 0.4-1.4, 0.2-1.0, and 0.97-4.63 pg/g Hb, respectively, are estimated from ETS exposure. These 4-ABP-Hb adduct levels from ETS exposure account for approximately 1-4% of the median levels reported for nonsmokers, explaining, in part, contradictory literature data on 4-ABP-Hb adduct levels in nonsmokers. No risk assessment of ETS or 4-ABP was conducted in this work, consequently the known health effects of ETS are neither confirmed or challenged and our conclusions are limited to the determination that ETS is not a major source of 4-ABP-Hb adduct levels in non-smokers. PMID:20433335

  5. Zonal distribution of protein-acetaldehyde adducts in the liver of rats fed alcohol for long periods.

    PubMed

    Lin, R C; Zhou, F C; Fillenwarth, M J; Lumeng, L

    1993-10-01

    Acetaldehyde, a highly reactive intermediate of alcohol metabolism, has been shown to form adducts with liver proteins in rats fed alcohol for long periods. In this report, the zonal distribution of liver protein-acetaldehyde adducts that formed in vivo was studied by means of histoimmunostaining. Rats were pair-fed alcohol-containing and alcohol-free AIN'76 liquid diets for 2 or 11 wk before they were killed and subjected to whole body perfusion with paraformaldehyde. Each liver was cut into 60-microns-thick slices. Slices were first treated with 10% hydrogen peroxide to eliminate endogenous peroxidase activity. They were then incubated sequentially with rabbit antihemocyanin-acetaldehyde adduct, goat antirabbit serum IgG and rabbit peroxidase-antiperoxidase complex. The liver slices were stained with diaminobenzidine and counterstained with methylgreen. In the livers of rats fed alcohol for 2 wk, peroxidase activity was evident in the perivenous zone but not the periportal zone. No staining was obtained when the primary antibody had been preabsorbed with immobilized hemocyanin-acetaldehyde adduct or if the liver slices were incubated with the unimmunized rabbit IgG. Slight staining of the perivenous zone was seen in the livers of control rats, presumably because of minimal protein-acetaldehyde adduct formation emanating from endogenous acetaldehyde. When rats were fed alcohol for longer periods (e.g., 11 wk), protein-acetaldehyde adducts were still seen predominantly in the perivenous zone, but the distribution pattern was more diffuse than that observed in the livers of rats fed alcohol for only 2 wk. More liver cells produced protein-acetaldehyde adducts when rats were fed the alcohol-containing diet supplemented with cyanamide.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Formation and repair of pyridyloxobutyl DNA adducts and their relationship to tumor yield in A/J mice

    PubMed Central

    Urban, Anna M.; Upadhyaya, Pramod; Cao, Qing; Peterson, Lisa A.

    2012-01-01

    The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a known human carcinogen. It generates methyl and pyridyloxobutyl DNA adducts. The role of the methyl DNA adducts has been well-established in the tumorigenic properties of NNK. However, the role of the pyridyloxobutyl DNA adducts is unclear. Four pyridyloxobutyl DNA adducts have been characterized: 7-[4-3-(pyridyl)-4-oxobut-1-yl]guanine (7-pobG), O2-[4-3-(pyridyl)-4-oxobut-1-yl]-cytodine (O2-pobC), O2-[4-3-(pyridyl)-4-oxobut-1yl]thymidine (O2-pobdT), and O6-[4-3-(pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-pobdG). Mutagenic O6-pobdG is thought to contribute to the tumorigenic properties of the pyridyloxobutylation pathway. It is repaired by O6-alkylguanine-DNA alkyltransferase (AGT). To explore the role of O6-pobdG formation and repair in the tumorigenic properties of NNK, A/J mice were given single or multiple doses of the model pyridyloxobutylating agent 4-(acetoxymethyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) in the presence or absence of the AGT depletor, O6-benzylguanine. Levels of the four pyridyloxobutyl DNA adducts were measured in the lung at 8, 48 or 96 h following treatment and compared to the lung tumorigenic activity of these treatments. AGT depletion had only a modest effect on the levels of O6-pobdG and did not increase tumor formation. Three pyridyloxobutyl DNA adducts, 7-pobG, O2-pobdT, and O6-pobdG, persisted in lung DNA at significant levels for up to 96 h post-treatment, suggesting that all three adducts may contribute to the tumorigenic properties of NNK. PMID:22928598

  7. High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts.

    PubMed

    Spencer-Beach, G G; Beach, A C; Gupta, R C

    1996-03-01

    32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible. PMID:8704930

  8. p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

    PubMed

    Lloyd, Daniel R; Hanawalt, Philip C

    2002-09-15

    Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the

  9. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase.

    PubMed

    Jiang, Wei; Murashko, Ekaterina A; Dubrovskii, Yaroslav A; Podolskaya, Ekaterina P; Babakov, Vladimir N; Mikler, John; Nachon, Florian; Masson, Patrick; Schopfer, Lawrence M; Lockridge, Oksana

    2013-08-15

    Exposure to nerve agents or organophosphorus (OP) pesticides can have life-threatening effects. Human plasma butyrylcholinesterase (BChE) inactivates these poisons by binding them to Ser198. After hours or days, these OP adducts acquire a negative charge by dealkylation in a process called aging. Our goal was to develop a method for enriching the aged adduct to facilitate detection of exposure. Human BChE inhibited by OP toxicants was incubated for 4 days to 6 years. Peptides produced by digestion with pepsin were enriched by binding to titanium oxide (TiO2) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It was found that with two exceptions, all aged OP adducts in peptide FGES198AGAAS were enriched by binding to Titansphere tips. Cresyl saligenin phosphate yielded two types of aged adduct, cresylphosphate and phosphate, but only the phosphate adduct bound to Titansphere. The nerve agent VR yielded no aged adduct, supporting crystal structure findings that the VR adduct on BChE does not age. The irreversible nature of aged OP adducts was demonstrated by the finding that after 6 years at room temperature in sterile pH 7.0 buffer, the adducts were still detectable. It was concluded that TiO2 microcolumns can be used to enrich aged OP-modified BChE peptide.

  10. Formation of 7-hydroxymethyl-12-methylbenz(a)anthracene-DNA adducts from 7,12-dimethylbenz(a)anthracene in mouse epidermis

    SciTech Connect

    DiGiovanni, J.; Nebzydoski, A.P.; Decina, P.C.

    1983-09-01

    The formation of DNA adducts from (/sup 3/H)-7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA) and (/sup 3/H)-7,12-dimethylbenz(a)anthracene (DMBA) in the epidermis of Sencar mice was analyzed. Comparison of Sephadex LH-20 chromatographic profiles of DNA samples isolated from mice treated with DMBA or 7-OHM-12-MBA suggested that the DMBA-treated animals contained DNA adduct(s) derived from the further metabolism of 7-OHM-12-MBA. Further analysis of DNA samples from DMBA-treated mice by high-pressure liquid chromatography demonstrated the presence of 5 DNA adducts which were chromatographically indistinguishable from the DNA adducts formed in 7-OHM-12-MBA-treated mice. Epidermal homogenates were utilized to catalyze the covalent binding of (/sup 3/H)DMBA and (/sup 3/H)-7-OHM-12-MBA to calf thymus DNA in vitro. Under conditions of limiting concentrations of (/sup 3/H)DMBA, the majority of the DNA adducts formed chromatographed in regions where 7-OHM-12-MBA-DNA adducts eluted. A major DMBA-DNA adduct formed in this in vitro system eluted with the same retention time as did the major 7-OHM-12-MBA-DNA adduct formed in mouse skin in vivo. These results when coupled with the in vivo data suggest that 7-OHM-12-MBA is an intermediate for at least some of the binding of DMBA to epidermal DNA in Sencar mice.

  11. Sugar Accumulation in Sugarcane

    PubMed Central

    Gayler, K. R.; Glasziou, K. T.

    1972-01-01

    The rate-limiting reaction for glucose uptake in storage tissue of sugarcane, Saccharum officinarum L., appears to be the movement of glucose across the boundary between the free space and the metabolic compartments. The mechanism for uptake of glucose across this boundary has been studied using 3-O-methyl glucose, an analogue of glucose which is not metabolized by sugar-cane tissue. This analogue is taken up by sugarcane storage tissue at a similar rate to glucose. Its rate of uptake follows Michaelis-Menten kinetics, Km = 1.9 mm, and it is competitively inhibited by glucose, Ki = 2 to 3 mm. Glucose uptake is similarly inhibited by 3-O-methyl glucose. Uptake of 3-O-methyl glucose is energy-dependent and does not appear to be the result of counterflow of glucose. It is concluded that glucose and 3-O-methyl glucose uptake across the boundary between the free space and the metabolic compartment in this tissue is mediated by an energy-dependent carrier system capable of accumulating the sugars against a concentration gradient. PMID:16658002

  12. TTX accumulation in pufferfish.

    PubMed

    Noguchi, Tamao; Arakawa, Osamu; Takatani, Tomohiro

    2006-03-01

    Tetrodotoxin (TTX) has been detected in a variety of animals. The finding of TTX in the trumpet shell Charonia sauliae strongly suggested that its origin was its food, a TTX-bearing starfish Astropecten polyacanthus. Since then, the food chain has been consistently implicated as the principal means of TTX intoxication. To identify the primary producer of TTX, intestinal bacteria isolated from several TTX-bearers were investigated for their TTX production. The results demonstrated that some of them could produce TTX. Thus the primary TTX producers in the sea are concluded to be marine bacteria. Subsequently, detritus feeders and zooplankton can be intoxicated with TTX through the food chain, or in conjunction with parasitism or symbiosis. The process followed by small carnivores, omnivores or scavengers, and by organisms higher up the food chain would result in the accumulation of higher concentrations of TTX. Finally, pufferfish at the top of the food chain are intoxicated with TTX. This hypothesis is supported by the fact that net cage and land cultures produce non-toxic pufferfish that can be made toxic by feeding with a TTX-containing diet.

  13. Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Aneba, Swida; Mateasik, Anton; Chorvat, Dusan; Comte, Blandine

    2013-06-01

    Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

  14. Mechanism of metabolic activation and DNA adduct formation by the human carcinogen diethylstilbestrol: The defining link to natural estrogens

    PubMed Central

    Saeed, Muhammad; Rogan, Eleanor

    2009-01-01

    Diethylstilbestrol (DES) is a human carcinogen, based on sufficient epidemiological evidence. DES is mainly metabolized to its catechol, 3′-hydroxyDES (3′-OH-DES), which can further oxidize to DES-3′,4′-quinone (DES-3′,4′-Q). Similarly to estradiol-3,4-quinone, the reaction of DES-3′,4′-Q with DNA would form the depurinating 3′-OH-DES-6′-N3Ade and 3′-OH-DES-6′-N7Gua adducts. To prove this hypothesis, synthesis of DES-3′,4′-Q by oxidation of 3′-OH-DES with Ag2O was tried; this failed due to instantaneous formation of a spiro-quinone. Oxidation of 3′-OH-DES by lactoperoxidase or tyrosinase in the presence of DNA led to the formation of 3′-OH-DES-6′-N3Ade and 3′-OH-DES-6′-N7Gua adducts. These adducts were tentatively identified by LC-MS/MS as 3′-OH-DES-6′-N3Ade, m/z = 418 [M+H]+, and 3′-OH-DES-6′-N7Gua, m/z = 434 [M+H]+. Demonstration of their structures derived from their oxidation by MnO2 to the DES quinone adducts and subsequent tautomerization to the dienestrol (DIES) catechol adducts, which are identical to the standard 3′-OH-DIES-6′-N3Ade, m/z = 416 [M+H]+, and 3′-OH-DIES-6′-N7Gua, m/z = 432 [M+H]+, adducts. The reaction of DIES-3′,4′-Q or lactoperoxidase-activated 3′-OH-DIES with DNA did not produce any depurinating adducts, due to the dienic chain being perpendicular to the phenyl planes, which impedes the intercalation of DIES into the DNA. Enzymic oxidation of 3′-OH-DES suggests that the catechol of DES intercalates into DNA and is then oxidized to its quinone to yield N3Ade and N7Gua adducts. These results suggest that the common denominator of tumor initiation by the synthetic estrogen DES and the natural estrogen estradiol is formation of their catechol quinones, which react with DNA to afford the depurinating N3Ade and N7Gua adducts. PMID:19089919

  15. N-Acetylcysteine blocks formation of cancer-initiating estrogen-DNA adducts in cells

    PubMed Central

    Zahid, Muhammad; Saeed, Muhammad; Ali, Mohammed F.; Rogan, Eleanor G.; Cavalieri, Ercole L.

    2010-01-01

    Catechol estrogens, especially 4-hydroxylated metabolites of 17β-estradiol (E2), are responsible for estrogen-induced carcinogenesis. 4-Hydroxyestradiol (4-OHE2), a major metabolite of E2 formed preferentially by cytochrome P-450 1B1, is oxidized to E2-3,4-quinone, which can react with DNA to yield the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua. The apurinic sites generated by the loss of these depurinating adducts induce mutations that could lead to cancer initiation. In the present study, we have evaluated the effects of N-acetycysteine (NAcCys) on the metabolism of two cell lines, MCF-10F (a normal human breast epithelial cell line) and E6 (a normal mouse mammary epithelial cell line), treated with 4-OHE2 or its reactive metabolite, E2-3,4-quinone. Extensive HPLC with electrochemical detection and UPLC-MS/MS analyses of the cell media demonstrated that the presence of NAcCys very efficiently shifted the estrogen metabolism towards protective methoxylation and conjugation pathways in multiple ways, while formation of depurinating DNA adducts was inhibited. Protection by NAcCys appears to be similar in both cell lines irrespective of their origin (human or mouse) or the presence of estrogen receptor-alpha. This finding suggests that NAcCys, a common dietary supplement, could be used as a potential chemopreventive agent to block the initial step in the genotoxicity caused by catechol estrogen quinones. PMID:20472053

  16. Prevention of estrogen-DNA adduct formation in MCF-10F cells by resveratrol

    PubMed Central

    Zahid, Muhammad; Gaikwad, Nilesh W.; Ali, Mohamed F.; Lu, Fang; Saeed, Muhammad; Yang, Li; Rogan, Eleanor G.; Cavalieri, Ercole L.

    2008-01-01

    Resveratrol (Resv), a natural occurring phytolexin present in grapes and other foods, possesses chemopreventive effects revealed by its striking modulation of diverse cellular events associated with tumor initiation, promotion and progression. Catechol estrogens generated in the metabolism of estrogens are oxidized to catechol quinones that react with DNA to form predominantly depurinating estrogen-DNA adducts. This event can generate the mutations responsible for cancer initiation. In this regard, Resv acts as both an antioxidant and an inducer of the phase II enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). In this report, we present the effects of Resv on the metabolism of estrogens in normal breast epithelial cells (MCF-10F) treated with 4-hydroxyestradiol (4-OHE2) or estradiol-3,4-quinone (E2-3,4-Q). Resv induced NQO1 in a dose- and time-dependent manner, but did not affect the expression of catechol-O-methyltransferase. Ultraperformance liquid chromatography/tandem mass spectrometry was used to determine the effects of Resv on estrogen metabolism. Preincubation of the cells with Resv for 48 h decreased the formation of depurinating estrogen-DNA adducts from 4-OHE2 or E2-3,4-Q and increased formation of methoxycatechol estrogens. When Resv was also present with the 4-OHE2 or E2-3,4-Q, even greater increases in methoxycatechol estrogens were observed, and the DNA adducts were undetectable. We conclude that Resv can protect breast cells from carcinogenic estrogen metabolites, suggesting that it could be used in breast cancer prevention. PMID:18423413

  17. Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

    SciTech Connect

    Gill, R.D.; Nettikumara, A.N.; DiGiovanni, J. ); Butterworth, B.E. )

    1991-01-01

    Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (B(a)P), ({plus minus}) 7{beta}-8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (({plus minus}) anti-BPDE), and ({plus minus}) 7{beta},8{alpha}-dihydroxy-9{beta},10{beta}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (({plus minus})syn-BPDE) to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present int he cell population when comparing similar compounds within the linear dose-response range of 0.005 {mu}g/ml-0.25 {mu}g/ml. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.

  18. Characterization of quinone derived protein adducts and their selective identification using redox cycling based chemiluminescence assay.

    PubMed

    Elgawish, Mohamed Saleh; Kishikawa, Naoya; Ohyama, Kaname; Kuroda, Naotaka

    2015-07-17

    The cytotoxic mechanism of many quinones has been correlated to covalent modification of cellular proteins. However, the identification of relevant proteins targets is essential but challenging goals. To better understand the quinones cytotoxic mechanism, human serum albumin (HSA) was incubated in vitro with different concentration of menadione (MQ). In this respect, the initial nucleophilic addition of proteins to quinone converts the conjugates to redox-cycling quinoproteins with altered conformation and secondary structure and extended life span than the short lived, free quinones. The conjugation of MQ with nucleophilic sites likewise, free cysteine as well as ɛ-amino group of lysine residue of HSA has been found to be in concentration dependent manner. The conventional methods for modified proteins identification in complex mixtures are complicated and time consuming. Herein, we describe a highly selective, sensitive, simple, and fast strategy for quinoproteins identification. The suggested strategy exploited the unique redox-cycling capability of quinoproteins in presence of a reductant, dithiothreitol (DTT), to generate reactive oxygen species (ROS) that gave sufficient chemiluminescence (CL) when mixed with luminol. The CL approach is highly selective and sensitive to detect the quinoproteins in ten-fold molar excess of native proteins without adduct enrichment. The approach was also coupled with gel filtration chromatography (GFC) and used to identify adducts in complex mixture of proteins in vitro as well as in rat plasma after MQ administration. Albumin was identified as the main protein in human and rat plasma forming adduct with MQ. Overall, the identification of quinoproteins will encourage further studies of toxicological impact of quinones on human health. PMID:26044383

  19. Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons.

    PubMed

    Klaene, Joshua J; Flarakos, Caroline; Glick, James; Barret, Jennifer T; Zarbl, Helmut; Vouros, Paul

    2016-03-25

    LC-MS using electrospray ionization is currently the method of choice in bio-organic analysis covering a wide range of applications in a broad spectrum of biological media. The technique is noted for its high sensitivity but one major limitation that hinders achievement of its optimal sensitivity is the signal suppression due to matrix inferences introduced by the presence of co-extracted compounds during the sample preparation procedure. The analysis of DNA adducts of common environmental carcinogens is particularly sensitive to such matrix effects as sample preparation is a multistep process which involves "contamination" of the sample due to the addition of enzymes and other reagents for digestion of the DNA in order to isolate the analyte(s). This problem is further exacerbated by the need to reach low levels of quantitation (LOQ in the ppb level) while also working with limited (2-5 μg) quantities of sample. We report here on the systematic investigation of ion signal suppression contributed by each individual step involved in the sample preparation associated with the analysis of DNA adducts of polycyclic aromatic hydrocarbon (PAH) using as model analyte BaP-dG, the deoxyguanosine (dG) adduct of benzo[a]pyrene (BaP). The individual matrix contribution of each one of these sources to analyte signal was systematically addressed as were any interactive effects. The information was used to develop a validated analytical protocol for the target biomarker at levels typically encountered in vivo using as little as 2 μg of DNA and applied to a dose response study using a metabolically competent cell line. PMID:26607319

  20. A nontargeted screening method for covalent DNA adducts and DNA modification selectivity using liquid chromatography-tandem mass spectrometry.

    PubMed

    Yao, Chunhe; Feng, Yong-Lai

    2016-10-01

    A method for nontargeted screening for covalent DNA adducts was developed using combination of neutral loss scan and product ion scan in a hybrid linear-ion-trap - triple quadrupole mass spectrometer system. DNA 2'-deoxynucleosides and adducts eluted from liquid chromatography were first analyzed in neutral loss mode to screen for the neutral loss of the deoxyribose moiety ([M+H-116](+)) from the protonated molecular ion ([M+H](+)). The product ion scan was subsequently used to elucidate the structures for the molecular ions observed from the peaks in the neutral loss scan chromatogram. The synthesized DNA adducts were used to evaluate the developed method by reaction of 20-mer DNA oligonucleotide with two direct agents respectively, specifically phenyl glycidyl ether and styrene-7,8-oxide. The modification selectivity of two compounds to the four nitrogenous bases on DNA sequence was also investigated in this study. The results showed that the two compounds had different modification selectivity to the four bases. Both compounds could modify all four nitrogenous bases (i.e. adenine, guanine, thymine, and cytosine) on DNA sequences to form various covalent DNA adducts. While phenyl glycidyl ether modified almost all of thymidine on DNA sequence, styrene-7,8-oxide, on the other hand, modified only a small portion of thymidine. The developed method proved possibly a potential tool for screening of unknown DNA adducts as exposure biomarkers of contaminants to human in the environment. PMID:27474284

  1. An abnormal N-heterocyclic carbene-carbon dioxide adduct from imidazolium acetate ionic liquids: the importance of basicity.

    PubMed

    Kelemen, Zsolt; Péter-Szabó, Barbara; Székely, Edit; Hollóczki, Oldamur; Firaha, Dzmitry S; Kirchner, Barbara; Nagy, József; Nyulászi, László

    2014-09-26

    In the reaction of 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc] ionic liquid with carbon dioxide at 125 °C and 10 MPa, not only the known N-heterocyclic carbene (NHC)-CO2 adduct I, but also isomeric aNHC-CO2 adducts II and III were obtained. The abnormal NHC-CO2 adducts are stabilized by the presence of the polarizing basic acetate anion, according to static DFT calculations and ab initio molecular dynamics studies. A further possible reaction pathway is facilitated by the high basicity of the system, deprotonating the initially formed NHC-CO2 adduct I, which can then be converted in the presence of the excess of CO2 to the more stable 2-deprotonated anionic abnormal NHC-CO2 adduct via the anionic imidazolium-2,4-dicarboxylate according to DFT calculations on model compounds. This suggests a generalizable pathway to abnormal NHC complex formation. PMID:25137312

  2. Mass spectrometry-based quantification of myocardial protein adducts with acrolein in an in vivo model of oxidative stress

    PubMed Central

    Wu, Jianyong; Stevens, Jan F.; Maier, Claudia S.

    2012-01-01

    Acrolein exposure leads to the formation of protein-acrolein adducts. Protein modification by acrolein has been associated with various chronic diseases including cardiovascular and neurodegenerative diseases. Here we report an analytical strategy that enables the quantification of Michael-type protein adducts of acrolein in mitochondrial proteome samples using liquid chromatography in combination with tandem mass spectrometry and selected ion monitoring (LC-MS/MS SRM) analysis. Our approach combines site-specific identification and relative quantification at the peptide level of protein–acrolein adducts in relation to the unmodified protein thiol pool. Treatment of 3-month old rats with CCl4, an established in vivo model of acute oxidative stress, resulted in significant increases in the ratios of distinct acrolein-adducted peptides to the corresponding unmodified thiol-peptides obtained from proteins that were isolated from cardiac mitochondria. The mitochondrial proteins that were found adducted by acrolein were malate dehydrogenase, NADH dehydrogenase [ubiquinone] flavoprotein 1, cytochrome c oxidase subunit VIb isoform 1, ATP synthase d chain, and ADP/ATP translocase 1. The findings indicate that protein modification by acrolein has potential value as an index of mitochondrial oxidative stress. PMID:21809440

  3. A new general pathway for synthesis of reference compounds of N-terminal valine-isocyanate adducts.

    PubMed

    Davies, Ronnie; Rydberg, Per; Westberg, Emelie; Motwani, Hitesh V; Johnstone, Erik; Törnqvist, Margareta

    2010-03-15

    Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization under the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.

  4. Dissociation and reduction of covalent β-lactoglobulin-quinone adducts by dithiothreitol, tris(2-carboxyethyl)phosphine, or sodium sulfite.

    PubMed

    Jongberg, Sisse; Lund, Marianne N; Otte, Jeanette

    2015-06-01

    Covalent protein-phenol adducts, generated by reaction of protein nucleophiles with quinones, have recently attracted increased attention because the interactions change the functionality and physicochemical properties of proteins in biological and food systems. The formation of such covalent adducts between β-lactoglobulin (β-LG) and the quinone of 4-methylcatechol, 4-methylbenzoquinone (4MBQ), and subsequent reduction by dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or sodium sulfite was investigated by mass spectrometry. The results showed that 19.0 ± 8.8% of β-LG reacted with 4MBQ when present in equimolar ratio at 20°C (pH 8.0) to yield the protein-phenol adduct (β-LG-Q). Following treatment with sulfite, DTT, or TCEP, 75, 68, or 36%, respectively, of the formed β-LG-Q adduct dissociated. Different reaction mechanisms were proposed for the reduction of β-LG and β-LG-Q by each of the reducing agents. These results show that on reductive sample preparation for analysis of protein samples, not only are protein polymers formed through oxidative disulfide bonds reduced into the individual protein constituents but also a large part of any protein-phenol adducts present will dissociate and, thus, give a false picture of the level of protein-protein interactions that have occurred in the sample. PMID:25700864

  5. RNA Oxidation Adducts 8-OHG and 8-OHA Change with Aβ42 Levels in Late-Stage Alzheimer's Disease

    PubMed Central

    Niedowicz, Dana M.; Dowling, Amy L. S.; Matveev, Sergey V.; LeVine, Harry; Lovell, Mark A.; Murphy, M. Paul

    2011-01-01

    While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain regions (Brodmann's Area 9, hippocampus, inferior parietal lobule, and the superior and middle temporal gyri), we found that the RNA adduct 8-hydroxyguanine (8-OHG) decreased, while 8-hydroxyadenine (8-OHA) increased in AD. The cerebellum, which is generally spared in AD, did not show disease related changes, and no RNA adducts correlated with the number of plaques or tangles. Multiple regression analysis revealed that SDS-soluble Aβ42 was the best predictor of changes in 8-OHG, while formic acid-soluble Aβ42 was the best predictor of changes in 8-OHA. This study indicates that although there is a connection between AD related neuropathology and RNA oxidation, this relationship is not straightforward. PMID:21949792

  6. Anion Effects on Sodium Ion and Acid Molecule Adduction to Protein Ions in Electrospray Ionization Mass Spectrometry

    PubMed Central

    Flick, Tawnya G.; Merenbloom, Samuel I.; Williams, Evan R.

    2012-01-01

    Gaseous protein–metal ion and protein–molecule complexes can be readily formed by electrospray ionization (ESI) from aqueous solutions containing proteins and millimolar concentrations of sodium salts of various anions. The extent of sodium and acid molecule adduction to multiply charged protein ions is inversely related and depends strongly on the proton affinity (PA) of the anion, with extensive sodium adduction occurring for anions with PA values greater than ~300 kcal·mol−1 and extensive acid molecule adduction occurring for anions with PA values less than 315 kcal·mol−1. The role of the anion on the extent of sodium and acid molecule adduction does not directly follow the Hofmeister series, suggesting that direct protein–ion interactions may not play a significant role in the observed effect of anions on protein structure in solution. These results indicate that salts with anions that have low PA values may be useful solution-phase additives to minimize nonspecific metal ion adduction in ESI experiments designed to identify specific protein-metal ion interactions. PMID:21952761

  7. Training multi-parameter gaits to reduce the knee adduction moment with data-driven models and haptic feedback.

    PubMed

    Shull, Pete B; Lurie, Kristen L; Cutkosky, Mark R; Besier, Thor F

    2011-05-17

    The purpose of this study was to evaluate gait retraining for reducing the knee adduction moment. Our primary objective was to determine whether subject-specific altered gaits aimed at reducing the knee adduction moment by 30% or more could be identified and adopted in a single session through haptic (touch) feedback training on multiple kinematic gait parameters. Nine healthy subjects performed gait retraining, in which data-driven models specific to each subject were determined through experimental trials and were used to train novel gaits involving a combination of kinematic changes to the tibia angle, foot progression and trunk sway angles. Wearable haptic devices were used on the back, knee and foot for real-time feedback. All subjects were able to adopt altered gaits requiring simultaneous changes to multiple kinematic parameters and reduced their knee adduction moments by 29-48%. Analysis of single parameter gait training showed that moving the knee medially by increasing tibia angle, increasing trunk sway and toeing in all reduced the first peak of the knee adduction moment with tibia angle changes having the most dramatic effect. These results suggest that individualized data-driven gait retraining may be a viable option for reducing the knee adduction moment as a treatment method for early-stage knee osteoarthritis patients with sufficient sensation, endurance and motor learning capabilities.

  8. Cytochrome P4501A induction and DNA adduct formation in glaucous gulls (Larus hyperboreus), fed with environmentally contaminated gull eggs.

    PubMed

    Østby, Lene; Gabrielsen, Geir Wing; Krøkje, Ase

    2005-11-01

    This study indicates that complex mixtures of pollutants found in the Arctic marine environment have genotoxic effects in glaucous gulls (Larus hyperboreus). DNA adducts were quantified, by the (32)P-postlabeling technique, in liver samples from gulls fed with hen eggs (controls) and from gulls fed with environmentally contaminated gull eggs (exposed). All birds were grown and fed under laboratory conditions. Hepatic homologues to mammalian cytochrome P4501A (CYP1A) proteins were also determined by Western blotting. DNA adducts were detected in all but one liver sample, but the exposed birds had a significantly increased level of DNA adducts relative to that of the controls. There was no clear significant correlation between the DNA adduct level and the level of organochlorine compounds (OCs) in blood. The level of CYP1A protein was significantly higher in the liver of exposed male gulls than in the liver of control males and positively correlated, with significance, to the level of OC compounds measured in blood. There was no significant correlation between the level of DNA adducts and the CYP1A protein content. PMID:16216630

  9. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.

  10. Covalent binding of benzo(a)pyrene diol epoxide to DNA of mouse skin: in vivo persistence of adducts formation

    SciTech Connect

    Shugart, L.

    1985-01-01

    In the first 9 d after topical application of a single dose of benzo(a)pyrene to the dorsal skin of C/sub 3/H mice, the half-lives of benzo(a)pyrene diol epoxide-DNA adducts and of DNA were determined to be approximately 5 d. These data indicate that, in proliferating mouse skin, benzo(a)pyrene diol epoxide-DNA lesions are not repaired, but are diluted from the genome at a rate equivalent to DNA turnover (i.e., replication versus degradation). Subsequent to this initial period, benzo(a)pyrene diol epoxide-DNA adduct removal continues, but at a much reduced rate. At 30 d posttreatment with benzo(a)pyrene, approximately 15% of the adducts are still detectable; however, their half-lives had increased to 30 d. Similar experiments with a hairless mouse showed that, although the amount of adduct formation was lower initially, the kinetics of aduct disappearance and persistence were essentially the same as found with the C/sub 3/H mouse. The data obtained in this work are consistent with the hypothesis that benzo(a)pyrene diol epoxide adducts persist in a subpopulation of skin cells long after their disappearance by DNA turnover would predict.

  11. Identification of two N{sup 2}-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene

    SciTech Connect

    Herreno-Saenz, D.; Evans, F.E.; Beland, F.A.

    1995-03-01

    1-Nitropyrene, the most abundant nitro-polycyclic aromatic hydrocarbon in the environment, is a known mammalian and bacterial mutagen and a tumorigen in animals. Early studies on DNA adduct characterization for 1-nitropyrene identified N-(deoxyguanosin-8-yl)-1-aminopyrene as the major product from the modification of calf thymus DNA with N-hydroxy-1-aminopyrene, the activated metabolite from nitroreduction of 1-nitropyrene. In this paper, we report the identification of two N{sup 2}-deoxyguanosinyl adducts, in addition to N-(deoxyguanosin-8-yl)-1-aminopyrene, formed from the reaction of N-hydroxy-1-aminopyrene, prepared in situ, with calf thymus DNA. These DNA adducts were identified as 6-(deoxyguanosin-N{sup 2}-yl)-1-aminopyrene and 8-(deoxyguanosin-N{sup 2}-yl)-1-aminopyrene. The two N{sup 2}-deoxyguanosinyl adducts were also identified in an ascorbic acid-catalyzed activation of 1-nitropyrene and in the mammary gland of female Sprague-Dawley rats administered 1-nitropyrene. The DNA adducts were also formed when 1-nitro