Unveiling the Timescale of the R-T Transition in Human Hemoglobin

Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place ∼ 2 μs after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-μs step observed with time-resolved optical spectroscopy corresponds to a small and localized structural change.     

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AαC), analysed by 32 P-HPLC

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by ³²P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AαC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AαC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AαC. The highest AαC–DNA adduct levels were found in liver (137 adducts/10⁸ normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N²-(deoxyguanin-8-yl)-2-amino-9H-pyrido[2,3-b]indole. Considering the abundance of AαC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AαC might be underestimated.     

Detection of lipid-lysine amide-type adduct as a marker of PUFA oxidation and its applications

Research into lipid peroxidation-induced protein modification has been ongoing for many years. Recent studies on lipo-oxidation shows the occurrence of another type of protein modification, amide-type adduct formation by lipid hydroperoxide, as well as classical aldehyde-derived protein modifications. The amide-type modifications can be either classified as alkylamide and carboxyalkylamide according to the formed structures. As an alkylamide-type adduct, Nε-(hexanoyl)lysine can be formed by the reaction of peroxidized n − 6 fatty acid with lysine. Nε-(propanoyl)lysine is considered to be generated from oxidation of n − 3 fatty acid with lysine. The generation pattern of both might be useful for classification of which fatty acids are more involved in oxidation in vivo. Since the alkylamide type-adducts are relatively stable and detectable from biological specimens like urine, these adducts, especially Nε-(hexanoyl)lysine, are used as reliable markers for not only oxidative stress evaluation but also development of functional food.     

Oxidative stress in neonates: evaluation using specific biomarkers

Increased oxidative stress has been implicated in pathogenesis of serious diseases in neonates. We measured urinary levels of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative DNA damage), acrolein-lysine adduct (a marker of lipid peroxidation and oxidative protein damage), and nitrite/nitrate (a marker of endogenous nitric oxide formation) in one-month-old neonates to examine the status of oxidative stress and its relationship to the degree of prematurity and clinical condition in neonates. Study subjects comprised three groups: healthy term neonates, clinically stable preterm neonates requiring no supplemental oxygen, and clinically sick preterm neonates requiring supplemental oxygen and ventilator support. Urinary levels of 8-hydroxy-2'-deoxyguanosine and acrolein-lysine adduct were significantly higher in sick preterm neonates than those of stable preterm and healthy term neonates. In the sick preterm group, neonates developing active retinopathy showed significantly higher levels of acrolein-lysine adduct than the other neonates without retinopathy. There were no significant differences in both urinary markers of oxidative stress between stable preterm and healthy term neonates. The urinary nitrite/nitrate levels were not significantly different among the three groups, suggesting no difference in endogenous nitric oxide formation. Collectively, these results provide evidence of augmentation of oxidative damage to DNA, lipids and proteins, especially in clinically sick preterm neonates.     

Development and application of oxidative stress biomarkers

Abstract

Oxidative stress may cause a wide variety of free radical reactions to produce deleterious modifications in membranes, proteins, enzymes, and DNA. Reactive Oxygen Species (ROS) generated by myeloperoxidase (MPO) can induce lipid peroxidation and also play an important role in the generation of reactive chlorinating and brominating species. As the universal biomarkers, chemical, and immunochemical approach on oxidatively modified and halogenated tyrosines has been carried out. As amido-type adduct biomarkers, chemical, and immunochemical evaluation of hexanoyl- and propanoyl-lysines, hexanoyl- and propanoyl-dopamines and phospholipids were prepared and developed for application of evaluation of novel antioxidative functional food factors. We have also involved in application of oxidatively modified DNAs such as 8-hydroxy- and 8-halogenated deoxyguanosines as the useful biomarkers for age-related diseases using both in vitro and in vivo systems. Application of these oxidative stress biomarkers for novel type of functional food development and recent approach for development of novel evaluation systems are also discussed.     

Crystallographic characterization of an exocyclic DNA adduct: 3,N4-etheno-2'-deoxycytidine in the dodecamer 5'-CGCGAATTepsilonCGCG-3'

Exocyclic DNA adducts are formed from metabolites of chemical carcinogens and have also been detected as endogenous lesions in human DNA. The exocyclic adduct 3,N(4)-etheno-2'-deoxycytidine (epsilon dC), positioned opposite deoxyguanosine in the B-form duplex of the dodecanucleotide d(CGCGAATTepsilonCGCG), has been crystallographically characterized at 1.8A resolution. This self-complementary oligomer crystallizes in space group P3(2)12, containing a single strand in the asymmetric unit. The crystal structure was solved by isomorphous replacement with the corresponding unmodified dodecamer structure. Exposure of both structures to identical crystal packing forces allows a detailed investigation of the influence of the exocyclic base adduct on the overall helical structure and local geometry. Structural changes are limited to the epsilon C:G and adjacent T:A and G:C base-pairs. The standard Watson-Crick base-pairing scheme, retained in the T:A and G:C base-pairs, is blocked by the etheno bridge in the epsilon C:G pair. In its place, a hydrogen bond involving O2 of epsilon C and N1 of G is present. Comparison with an epsilon dC-containing NMR structure confirms the general conformation reported for epsilon C:G, including the hydrogen bonding features. Superposition with the crystal structure of a DNA duplex containing a T:G wobble pair shows similar structural changes imposed by both mismatches. Evaluation of the hydration shell of the duplex with bond valence calculations reveals two sodium ions in the crystal.     

2-Hydrazinobenzothiazole-based etheno-adduct repair protocol (HERP): A method for quantitative determination of direct repair of etheno-bases

Etheno-DNA adducts are mutagenic and lead to genomic instability. Enzymes belonging to Fe(II)/2-oxoglutarate-dependent dioxygenase family repair etheno-DNA adducts by directly removing alkyl chain as glyoxal. Presently there is no simple method to assess repair reaction of etheno-adducts. We have developed a rapid and sensitive assay for studying etheno-DNA adduct repair by Fe(II)/2-oxoglutarate-dependent dioxygenases. Using AlkB as model Fe(II)/2-oxoglutarate-dependent dioxygenases, we performed in vitro repair of etheno-adducts containing DNA and detected glyoxal by reacting with 2-hydrazinobenzothiazole which forms complex yellow color compound with distinct absorption spectrum with a peak absorption at 365 nm. We refer this method as 2-hydrazinobenzothiazole-based etheno-adduct repair protocol or HERP. Our novel approach for determining repair of etheno-adducts containing DNA overcomes several drawbacks of currently available radioisotope-based assay.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the P-postlabeling technique

Using the ³²P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with β-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by ³²P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C₁₁---C₁₂ bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: blocking its artifact formation with glutathione

Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N2-propanodeoxyguanosine adducts (Acr-dG). Studies demonstrate that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the study described here, a quantitative 32P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1 fmol. It was found that Acr-dG can form as an artifact during the assay. Evidence obtained from mass spectrometry indicates that the Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, Acr-dG was detected as a contaminant in the commercial dG and dT 3'-monophosphate samples. Finally, this method was used to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship.     

Structural and functional characterization of BaiA,an enzyme involved in secondary bile acid synthesis in human gut microbe

Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo‐ and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2′‐phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (k_cat/K_M) of NADP⁺ at least an order of magnitude lower than NAD⁺. Substitution of Glu42 with Ala improved specificity toward NADP⁺ by 10‐fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide‐hydroxyl ion (NAD⁺‐OH^?) adduct contraposing previously reported adducts. The OH^? of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2′‐hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD⁺‐OH^? adduct in proton relay instead of hydride transfer as noted for previous adducts. Proteins 2014; 82:216–229. © 2013 Wiley Periodicals, Inc.