Effects of Polycyclic Aromatic Hydrocarbon Adducts with Deoxyguanosine and Deoxyadenosine in vivo and in vitro

Reactive metabolites from non-planar polycyclic aromatic hydrocarbon carcinogens react extensively with both deoxyadenosine and deoxyguanosine in DNA whereas those from planar molecules react predominantly only with deoxyguanosine. In vitro studies with single adducts in oligonucleotides showed that both types of adduct blocked primer extension and that the limited amount of nucleotide addition opposite the adduct varied with the polymerase, the sequence context of the adduct and the chemical structure of the adduct. When these same single adduct containing-oligonucleotides were introduced into a single-stranded vector that was allowed to replicate in Escherichia coli, the major events observed were blockage of replication, insertion of the correct nucleotide (i.e. T opposite an A adduct and C opposite a G adduct), and insertion of A opposite the adduct. In mouse skin, benzo[c]phenanthrene 4S,3R-dihydrodiol 2S,1R-epoxide initiated substantially more tumors per DNA adduct formed than did the other three isomers. This isomer generates mostly adenine adducts in DNA, suggesting that, in this case, adenine adducts are intrinsically more tumorigenic than guanine adducts.     

The Photocycloaddition of Olefins to Unsaturated 3-Ketosteroids

Photoadditions of ethylene and of cyclopentene to Δ⁴ - and Δ^4,6-3-ketosteroids gave mixtures of cis -4α,5α and trans -4α. 5β adducts. In the case of cyclopentene and Δ^4,6-androstadiene-3-one-17β-ol propionate, only the trans adduct was observed. Cis -4β,5β adducts were obtained by base-catalyzed isomerization of the trans adducts. The ratio of cis:trans adducts varied considerably with solvent, temperature, and olefin concentration in steroid reactions but not in reactions with cyclohexenone. The different types of adducts exhibited markedly different ORD curves in agreement with the structures assigned. Carbonyl maxima of trans adducts appeared at shorter wavelengths than maxima of cis adducts in both infrared and ultraviolet spectra. The infrared shifts provide a convenient method for differentiating between cis and trans fused isomers.     

DNA Polymerase Action on Oligonucleotide Templates from Human Ha-ras Protooncogene Containing N6-Deoxyadenosine Adducts Derived from Trans Addition of (+)- and (-)-anti-Benzo[c]phenanthrene-3,4-dihydrodiol 1,2-epoxides at Codon 61

In the present work we have used a DNA polymerase assay to investigate the primer extension with T7 DNA polymerase (Sequenase 2.0) and the Klenow fragment of Escherichia coli DNA polymerase I (exo ^? KF) on chemically synthesized 21mer templates representing partial sequences of the human Ha-ras protooncogene with site-specifically positioned trans-N ⁶-dA adducts of (-)- (adduct 1) and (+)-anti-benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides (adduct 2) at codon 61 (CA?G; A? indicates the adducted position). With Sequenase 2.0 a complete block of primer extension opposite both adduct 1 and 2 was noted using a 10mer primer reaching the (n-1)-position of the adduct. A detailed analysis revealed that Sequenase 2.0 exclusively allowed the misincorporation of dAMP opposite both adducts. In contrast, exo ^? KF allowed a significant full-length extension of the primer beyond adduct 2, whereas this process occured only to a very minor extent for adduct 1. Correspondingly, a more intensive block of the polymerization reaction opposite adduct 1 was seen. Analysis of the arrest bands revealed that dAMP was preferentially incorporated opposite to adduct 1; also incorporation of dGMP was observed, but to a minor extent. Similarly, dAMP was also incorporated opposite to adduct 2 along with dTMP, the latter indicating a correct base pairing. Additional experiments with exo ^? KF using different 11mer primers containing each of the four nucleotides opposite the adduct suggest that the primer extension beyond adduct 2 observed with the 10mer primer is an error-free process.     

Effect of Dietary Restriction on DNA-Adduct Formation of Benzo[a]pyrene and 2-Acetylaminofluorene in Mouse Liver

Dietary restriction (DR) alters hepatic drug metabolizing enzyme activities that affect the metabolic activation of xenobiotics. Previously, we have studied the effect of DR on the formation of benzo[a]pyrene (BaP)-DNA adducts in male Fischer 344 rats. In this study, the effects of DR on the formation of specific BaP-DNA adducts in mouse livers were investigated. In addition, 2-acetylaminofluorene (2-AAF) modified DNA adducts in livers of mice and rats were also examined. DR reduced the body and liver weights of male B6C3F₁ mice. Both the total [³H]BaP-DNA binding and the specific BaP-DNA adduct, N²-dG-BaP, detected by ³²P-postlabeling technique, were found to be greater in DR mice than in animals fed al libitum (AL). The formation of the 2-AAF-DNA adduct, AF-C8-dG, was not affected by DR. Similar results were obtained from the in vitro DNA adduct formation of these two carcinogens mediated by rat and mouse liver microsomes. The effect of DR on the formation of BaP- and 2-AAF-modified DNA adducts is discussed.     

Synthesis of Stereoisomeric N 6-Deoxyadenosine Adducts of syn- and anti- Dihydrodiol Epoxides of Benzo[a]pyrene and Their Incorporation into 18-mer DNA Sequences from Human Ha-ras Protooncogene

Oligonucleotide sequences synthesized with specifically positioned and structurally defined adducts of dihydrodiol epoxides (DE) of polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) are useful tools to study in detail the solution structure of corresponding duplexes by NMR techniques as well as the interaction of a single adduct with DNA polymerases. Here we report the successful incorporation of trans-N ⁶-dA-B[a]PDE adducts derived from the syn- and anti-diastereomers of B[a]PDE into 18-mer oligonucleotides representing partial human Ha-ras sequences surrounding codon 61 (CAG). The key step in our approach is a nucleophilic displacement reaction of a deoxyinosine derivative activated at the 6-position by a sulfonate group with a racemic aminotriol providing a regio- and stereospecific synthesis of the N ⁶-dA adducts. The aminotriol precursors are prepared by direct aminolysis of the DE's or by a stereoselective opening of the oxirane ring with sodium azide followed by reduction. The fully protected diastereomeric trans-N ⁶-dA-B[a]PDE adducts were separated by preparative HPLC and subsequently transformed into the corresponding phosphoramidites. The synthesis of four 18-mers was performed on a DNA synthesizer incorporating in each sequence [d(5′-GGC·CA*G·GAG·GAG·TAC·AGC-3′)] a single dA adduct (A*) at Codon 61 using standard phosphoramidite chemistry. After extensive purification of the 18-mers by reverse phase HPLC and analysis by PAGE, the presence of the trans-N⁶ -dA-B[a]PDE adducts in the oligonucleotides was confirmed by fluorescence spectroscopy.     

A novel method for color determination of edible oils in L*a*b* format

A simple method that uses visible spectrophotometer data and an artificial neural network (ANN) was developed to determine edible oil color based on the L*a*b* format. The 100 oil samples consisted of nine pure oils, a sesame oil blend and three heated oils. Binary, ternary and quaternary mixtures of these 13 oils in different ratios were prepared, and absorbance values of the samples were measured in the visible region (380–700 nm). The absorbance values at wavelengths of 416, 456, 483, 537, 611 and 672 nm were used to train, validate and test the network. Strong correlations between the instrumental L*a*b*ΔE and the estimated L*a*b*ΔE were found for the test samples, with correlation coefficients (R²) of 0.989, 0.984, 0.996 and 0.992 for L*, a*, b*, and ΔE, respectively. The effects of number and combination of the wavelengths used for training of the ANN on the estimation capability of the network for the test samples were also investigated. Although a good agreement, average R² of 0.991– 0 993 for L*a*b*, was obtained for combinations composed of three to six wavelengths with 483 and 537 nm in common, the best R² value was obtained when all six wavelengths were used to train the ANN. The developed method is objective, cost effective and simple, and allows the color measurement with a basic visible spectrophotometer and disposable cuvettes.     

DNA Adducts Formed in vitro from Fractionated Extract of Urban Air Particles

This study is a part of an ongoing interdisciplinary project on the health effects of air pollution (Teplice Program) in the highly polluted district of Teplice, Northern Bohemia. In our previous studies we found the relationship between DNA adduct levels detected in white blood cells of selected Teplice population and personal exposure to PAH associated with respirable particles. In this pilot study we used ³²P-postlabeling assay for DNA adduct detection in an in vitro model system for evaluation of genotoxic activity of fractionated urban air extractable organic matter (EOM). To identify some of the specific DNA adducts formed we coupled TLC with HPLC analysis of labeled adducts. The urban air particles were collected by high-volume sampler during January-March 1992 in Teplice. EOM was extracted by dichlormethane (DCM) and crude extract was fractionated into five fractions to obtain a gross partition of different chemical classes. Fractions were incubated with calf thymus DNA (dose 100 μg/ml incubate) under oxidative and reductive conditions using two metabolic activation system: 1) an oxidative rat liver S9 system (S9) and 2) a reductive xanthine oxidase catalyzed system (XO). The different DNA adduct patterns and levels were determined using S9 and XO-mediated metabolism followed by postlabeling with both nuclease P1 and butanol extraction enrichment procedures for all fractions examined. The moderately polar fraction (DCM) contained over 50% of the total DNA adduct forming activity both without and with S9 activation. The highly polar fraction (methanol) contained about 60 % of the DNA adduct forming activity under reductive conditions. Some of the main distinct DNA adducts obtained with S9 and XO mediated metabolism were tentatively identified by HPLC comparing with standards of PAH- and nitro-PAH DNA adducts.     

Nematicidal sphingolipids from the freshwater fungus Paraniesslia sp. YMF1.01400

A new ( 1 ) and a known ( 2 ) sphingolipid were isolated from mycelial cultures of Paraniesslia sp. YMF1.01400. The structures of these two isolates were elucidated as (2S,2'R,3R,3'E,4E,8E)‐1‐O‐(β‐D ‐glucopyranosyl)‐3‐hydroxyl‐2‐[N‐2'‐hydroxyl‐3'‐eico‐sadecenoyl]amino‐9‐methyl‐4,8‐octadecadiene ( 1 ) and (2S,2'R,3R,3'E,4E,8E)‐1‐O‐(β‐D ‐glucopyranosyl)‐3‐hydroxyl‐2‐[N‐2'‐hydroxyl‐3'‐octadecenoyl]amino‐9‐methyl‐4,8‐octadecadiene ( 2 ) by spectroscopic and chemical methods. In in vitro tests, both compounds showed moderate nematicidal activities against Bursaphelenchus xylophilus. This is the first report of secondary metabolites in a genus Paraniesslia aquatic fungus.     

Isolation and structure of glucosylceramides from the starfish Cosmasterias lurida

From the water-insoluble lipid fraction of the methylene chloride/methanol extract of the starfish Cosmasterias lurida, two new glucosylceramides together with a known glucosylceramide, ophidiacerebroside E, were isolated by chromatographic procedures and characterized by spectroscopic (¹H and ¹³C nuclear magnetic resonance, mass spectrometry) methods. The new compounds were identified as (2S, 3R, 4E, 8E, 10E)-1-(β-d-glucopyranosyloxy)-3-hydroxy-2-[(R)-2-hydroxyheptadecanoyl)amino]-9-methyl-4,8,10-octadecatriene (3) and (2S,3R,4E,8E,10E)-1-(β-d-glucopyranosyloxy)-3-hydroxy-2-[(R)-2-hydroxyoctadecanoyl)amino]-9-methyl-4,8,10-octadecatriene (4).     

Density,refractive index,excess molar volumes and deviations in molar refraction at 298.15 K for binary and ternary mixtures of DIPE (OR TAME) + 1‐methanol (or 1‐propanol) + trihexyltetra‐decylphosphonium bis (2,4,4‐trimethylpentyl) phosphinate

The excess molar volumes (V^E) and the deviations in molar refraction (ΔR) at 298.15 K were determined for the binary systems {diisopropyl ether (DIPE) + 1‐propanol}, {Tert‐amyl methyl ether (TAME) + methanol}, {DIPE + trihexyltetradecylphosphonium bis(2,4,4‐trimethylpentyl)phosphinate ([P_666,14][TMPP])}, {TAME + [P_666,14][TMPP]}, {methanol + [P_666,14][TMPP]} and {1‐propanol + [P_666,14][TMPP]} using a digital vibrating‐tube densimeter and a precision digital refractometer. The V^E and ΔR were correlated with the Redlich–Kister equation for binary systems. In addition, the ternary V^E and ΔR data at 298.15 K were predicted for the ternary systems {DIPE + 1‐propanol + [P_666,14][TMPP]} and {TAME + methanol + [P_666,14][TMPP]} by using the binary contribution model of Radojkovi? with correlated sub‐binary Redlich–Kister parameters. © 2011 Canadian Society for Chemical Engineering     

POLYCYCLIC AROMATIC HYDROCARBONS OF DIESEL AND GASOLINE EXHAUST AND DNA ADDUCT DETECTION IN CALF THYMUS DNA AND LYMPHOCYTE DNA OF WORKERS EXPOSED TO DIESEL EXHAUST

Particles present in urban air pollution are mainly derived from diesel- and gasoline-fueled vehicles. Exhaust emission is able to cause several health effects in humans including mutagenicity and carcinogenicity. Polycyclic aromatic hydrocarbons (PAHs) were measured in diesel and gasoline particulate extracts and DNA binding to CT DNA (±S9 and ±XO) was investigated. A large difference in content of 14 PAHs in diesel and gasoline extracts was observed, showing higher concentration of 14 PAHs, 6 carcinogenic PAHs, benzo[a]pyrene (B[a]P) in diesel than in gasoline extracts. Selected PAH standards of B[a]P, benzo[c]fluoranthene (B[c]F), and 3-nitrobenzanthrone (NBA) were used in ³²P-postlabeling/polyacrylamide gel electrophoresis (PAGE) technique to identify CT DNA adducts formed by diesel particulate extracts. CT DNA adduct formation was higher for diesel extracts in comparison with gasoline extracts; however, no clear origin of DNA adducts derived from B[c]F-, 3-NBA-, B[a]P was detected. ³²P-postlabeling/PAGE was a useful assay for analyzing and identifying PAH-DNA adducts. Occupational exposure to particulate and volatile PAH concentrations were evaluated using personal air samples and lymphocyte DNA adducts as markers of exposure. Overall air PAH concentrations were low in all eight workplaces, consisting of 97% of vapor phase compounds. DNA adducts analyzed by ³²P-postlabeling assay were compared between the butanol and nuclease P1 enrichment procedures. Only in winter samples of exposed workers, butanol extraction revealed significantly higher adduct levels in comparison with those of control persons. No differences in adduct levels between exposed and control persons in summer were detected by using either butanol extraction or nuclease P1 treatment. Total concentrations of particulate and volatile PAHs measured in eight workplaces in winter showed a significant correlation with total DNA adducts analyzed in workers' lymphocytes (r = 0.852N = 8, p = .007).     

Studies with inorganic precipitate membranes—XII: Consideration of membrane field strength and energetics of permeation

Electrolytic transfort processes occuring across parchment supported membranes have been described by Nernst Planck flux equation taking into account the membrane resistance R_m, membrane potential E_metc.E_m values for various electrolytes display very interesting phenomena. In the case of 1:1 electrolyte the E_m values are all positive, while in the case of (2:1) and (3:1) electrolytes surface charge reversal takes place. The diffusion rate sequence and selectivity of the membrane for different uni- bi- and trivalent cations was found to be primarily dependent on the difference in the hydration energies of counter ions in the external solution. On the basis of Eisenman-Sherry theory the diffusion rate sequence of alkali metal cations point towards the weak field strength of the fixed charge groups. Various thermodynamic parameters, ΔH^≠, ΔF^≠ and ΔS^≠ were evaluated by applying the theory of absolute reactions rates to the diffusion process through parchment supported membranes. The values of ΔS^≠ were found to be negative, indicating that diffusion takes place with partial immobilization in the membrane phase. The relative partial immobility was found to increase with increase in the valence of the ions constituting the electrolyte. A formal relation between ΔH_hydration, ΔF_hydration and ΔS_hydration of cations with the corresponding values of ΔH^≠, ΔF^≠ and ΔS^≠ for diffusion, was also found to exist for these membranes.     

Structure and Energy Spectra of a Class of Polybenzenoid Clar's Hydrocarbons and 1-D Polymers

The energy spectra of a class of 2-D polybenzenoid Clar 's hydrocarbons with a large number N(N ～ 10⁴) of carbon atoms is studied theoretically. It is shown that at the asymptotic case N ∞) the energy gap (EG) δE (N ∞) is different from zero, i.e., the π-systems should possess semiconductor properties. The results for the EG ΔE (N ∞) ≠ 0 of the hydrocarbons are in qualitative agreement with the results for the EG calculated for a class of 1-D ladder polymers having the same edge structure as the hydrocarbons. With increasing the number (M) of the π-centers of the elementary unit of the polymers, the band gap ΔE(M ∞) approaches also to a value different from zero. The quantitative results on the equilbria geometries of the hydrocarbons and the polymers correspond to Clar 's qualitative characterization of benzenoids composed of disjoint “π-sextets”. The energetics of the hydrocarbons with different types of defects and the corresponding Tamm and Frenkel states were also investigated.     

Influence of pregrafting cotton fabrics on kinetics of DMEU finishing

Cotton fabrics were pregrafted with a mixture of N-methylolacrylamide and methacrylic acid at mixing molar ratio of 4/6. The influence of pregrafting on the kinetics of finishing with 1,3-dimethylolethylene urea was studied. The results shows that pregrafting can increase the rate constants. Values of E_a ΔH^*, ΔS^*, and ΔG^* suggest that the pregrafting of cotton fabric not only affects the reaction action state, but also is beneficial for the reaction between cellulose and the finishing agent. © 1996 John Wiley & Sons, Inc.     

Impact of Site-Specific Benzo[a]Pyrene Diol Epoxide-dG Lesions at or near Single/Double-Strand DNA Junctions on DNA Bending

Adducts derived from the binding of the (+)-7R,8S,9S,10R and (?)-7S,8R,9R,10S enantiomers of r7,t8-dihydrodiol-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to 2′-deoxyguanosine residues in DNA are known to induce mutations due to error-prone DNA replication. Because the conformational properties of these lesions may be important in these phenomena, we have examined the effects of the stereoisomeric (+)- and (?)-trans-anti-[BP]-N ²-dG lesions positioned site-specifically at or near primer/template oligonucleotide junctions on DNA bending using high resolution gel electrophoresis. Remarkable differences in electrophoretic mobilities μ are observed in the two adducts derived from the tumorigenic (+)-anti-BPDE, and the non-tumorigenic (?)-anti-BPDE enantiomer. With the (+)-trans lesion positioned on the template strand adjacent to the 3′-end of the primer strand, a remarkable decrease in μ is observed. This suggests the existence of a bend at the single strand-double strand junction. Only modest decreases in μ are observed in the case of the (?)-trans lesion, or when the 3′-end is opposite to, or more distant from the lesion site. These observations are discussed in terms of the known NMR solution structures of these lesions in the same sequence context, and the properties of primer/template DNA in polymerases.     

Thermal and spectroscopic studies of poly(N‐vinyl pyrrolidone)/poly(vinyl alcohol) blend films

Poly(N‐vinyl pyrrolidone) (PVP) and poly (vinyl alcohol) (PVA) homopolymers and their blended samples with different compositions were prepared using cast technique and subjected to X‐ray diffraction (XRD) measurements, infrared (IR) spectroscopy, ultraviolet/visible spectroscopy, and thermogravimetric analysis (TGA). XRD patterns of homopolymers and their blended samples indicated that blending amorphous materials, such as PVP, with semicrystalline polymer, such as PVA, gives rise to an amorphous structure with two halo peaks at positions identical to those found in pure PVP. Identification of structure and assignments of the most evident IR ‐ absorption bands of PVP and PVA as well as their blends in the range 400–2000 cm^?1 were studied. UV–vis spectra were used to study absorption spectra and estimate the values of absorption edge, E_g, and band tail, E_e, for all samples. Making use of Coats‐Redfern relation, thermogravimetric (TG) data allowed the calculation of the values of some thermodynamic parameters, such as activation energy E, entropy ΔS^#, enthalpy ΔH, and free energy of activation ΔG^# for different decomposition steps in the samples under investigation. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

O6‐Alkylguanine‐DNA Alkyltransferase‐Mediated Repair of O4‐Alkylated 2′‐Deoxyuridines

O⁶‐Alkylguanine‐DNA alkyltransferases (AGTs) are responsible for the removal of O⁶‐alkyl 2′‐deoxyguanosine (dG) and O⁴‐alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O⁶‐alkylguanine‐DNA‐alkyltransferase) protein, which can repair a range of O⁴‐alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O⁴‐alkyl 2′‐deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada‐C and OGT), human AGT, and an OGT/hAGT chimera to remove O⁴‐methyl and larger adducts (4‐hydroxybutyl and 7‐hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O⁴‐methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O⁴ atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct‐containing oligonucleotides and the amounts of repair measured.     

Metabolic Activation of Dibenzo[a,l]Pyrene by Cytochrome P450 Enzymes to Stable DNA Adducts Occurs Exclusively Through the Formation of the (−)-trans−(11R, 12R)-Diol

At low doses, the carcinogenic hexacyclic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) forms DNA adducts in human MCF-7 cells exclusively through formation of the (–)-anti?(11R, 12S)-diol (13S, 14R)-epoxide (DB[a,l]PDE) via its metabolic precursor, the (–)-(11R, 12R)-diol. The same result was obtained by exposure of V79 cells stably expressing human cytochrome P450 (P450) 1B1. Although several other metabolites such as the 7-phenol and a 11, 12-diol phenol are formed, no other DNA adducts are detectable after exposure to 0.1 μM DB[a,l]P. Exposure to 1 μM DB[a,l]P leads to the formation of low levels of (+)-syn?DB[a,l]P-DE-DNA adducts through intermediate generation of the (+)-(11S, 12S)-diol. In contrast, treatment of P450 1A1-expressing V79 cells results also in the formation of several polar DNA adducts. Incubation of the trans?8,9-diol and trans?11, 12-enantiomers of DB[a,l]P demonstrated that all polar DNA adducts detected in 1A1-expressing cells resulted from intermediate formation of the (–)-trans?11, 12-diol. Although the K-region trans?8,9-diol is metabolically converted to several diol phenols and bis?diols, this compound does not contribute to the strong DNA binding or cytotoxicity observed after exposure of P450 1A1- and 1B1-expressing cells to the parent hydrocarbon.     

Three‐dimensional color prediction modeling of single‐ and double‐layered woven fabrics

In this research, the three‐dimensional structural and colorimetric modeling of three‐dimensional woven fabrics was conducted for accurate color predictions. One‐hundred forty single‐ and double‐layered woven samples in a wide range of colors were produced. With the consideration of their three‐dimensional structural parameters, three‐dimensional color prediction models, K/S‐, R‐, and L*a*b*‐based models, were developed through the optimization of previous two‐dimensional models which have been reported to be the three most accurate models for single‐layered woven structures. The accuracy of the new three‐dimensional models was evaluated by calculating the color differences ΔL*, ΔC*, Δh°, and ΔE_CMC(2:1) between the measured and the predicted colors of the samples, and then the error values were compared to those of the two‐dimensional models. As a result, there has been an overall improvement in color predictions of all models with a decrease in ΔE_CMC(2:1) from 10.30 to 5.25 units on average after the three‐dimensional modeling.