Fraction Of Adducts Research Articles

Benzo[a]pyrene diol epoxide adduct formation in mouse and human hemoglobin: physicochemical basis for dosimetry

The interactions of human hemoglobin (hHb) with the anti-diol epoxide of benzo[a]pyrene (aBaPDE) and with the corresponding tetrols formed by hydrolysis of the epoxide were investigated with the aim of characterizing the covalent adducts formed by reaction of the epoxide with the protein. The major product (80% of the total) was determined to be an ester resulting from oxirane ring opening by one or several (unidentified) carboxylate group(s). Minor products were characterized as adducts formed by reaction with amino or heterocyclic nitrogen by comparison of their UV spectra with those of model compounds. There was no evidence for reaction with cysteine. Formation of ester adducts by aBaPDE with mouse hemoglobin (mHb) following administration of BaP to mice was also investigated. It was found that esters constituted the majority of the adducts formed by aBaPDE and a substantial fraction of the total adducts formed. The esters formed by mHb were significantly less stable than those formed by hHb, both in vivo and in vitro. The instability of mHb ester adducts is believed by the responsible for differences among previous descriptions of the in vivo binding of BaP to mHb.

Radiation-induced Crosslinking between Poly(deoxyadenylic-deoxythymidylic Acid) and Tripeptides Containing Aromatic Residues

OH.-induced covalent peptide-nucleotide adducts have been isolated by reverse-phase chromatography from the enzymic hydrolyzates of gamma-ray irradiated solutions containing double-stranded poly(deoxyadenylic-deoxythymidylic acid) and one of the tripeptides, lysyl-tryptophyl-lysine or lysyl-tyrosyl-lysine. Numerous compounds were formed, resulting presumably from different modes of radical addition. All isomers appeared to have the same general structure peptide-d(ApTpA), based mostly on double-labelling experiments of bases and phosphate groups in DNA. The major adduct fraction obtained from Lys-Trp-Lys and poly(dA-dT) was purified to homogeneity by sequential reverse-phase and ion-exchange chromatography, and characterized spectrally. The pattern of acid and alkaline hydrolysis suggests that thymine is the site of peptide-nucleotide binding in this particular adduct fraction.

Repair synthesis by human cell extracts in DNA damaged bycis- andtrans-diamminedichloroplatinum(II)

DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA.

Heat treatment of vegetable oils I. Isolation of the cyclic fatty acid monomers from heated sunflower and linseed oils

AbstractLinseed and sunflower oils were heated at 275 C for 12 hr under nitrogen. The sunflower oil was also heated in a commercial fryer at 200 C for 48 hr using a 2‐hr daily cycle. The cyclic fatty acid monomers (CFAM) formed during the heat treatment of the linseed oil were isolated by a combination of saponification, esterification, column chromatography on silicic acid and urea fractionation. The isolated CFAM fraction was 99% pure, the balance being some 12:2ω6. Another step was necessary to isolate the CFAM from heated sunflower oils. The urea adduct fractionation resulted in the isolation of a nonurea adduct fraction which contained a mixture of CFAM and 18:2ω6. These were further separated using high performance liquid chromatography (HPLC) on a C18 reverse phase column. Each fraction was analyzed by gas liquid chromatography and hydrogenated to determine the content of the C18 straight chain fatty acids.

32P-postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin.

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the adducts; 3-OH-DBC and the other two phenolic metabolites investigated play a minor role, if any, in the formation of DBC-DNA adducts in skin; metabolic activation of DBC to DNA-binding compounds in liver and skin appears to follow pathways that are different in terms of both the chemical nature and the amount of the adducts formed; and DBC and 3-OH-DBC exhibit a strong preference for liver versus skin DNA.

Differential Modification of Hemoglobin Chains by Acetaldehyde

Acetaldehyde-hemoglobin adducts have been suggested as potential markers for alcohol consumption. These adducts were formed in vitro with [14C]acetaldehyde and separated into hemoglobin subunits by cation-exchange chromatography to examine the relative modification of the alpha- and beta-chains. The effect of varying concentrations of acetaldehyde on the relative amounts of polypeptide adducts and on the specific radioactivities of undissociated hemoglobin (Hb) following reaction with hydroxymercurybenzoate (HMB) was also studied. There were linear relationships (P less than 0.05) between increasing levels of [14C]acetaldehyde (0.0, 0.1, 0.2, 0.5 mM) and the radioactivities of the alpha- and one of the two beta-chain adducts (22, 25, 53 dpm/mg Hb and 151, 272, 626 dpm/mg Hb, respectively). Increases in radioactivities of a minor unidentified hemoglobin adduct fraction were also observed. The ratios of specific radioactivities of beta-to alpha-chain (8.8 +/- 1.2 SEM) did not vary with the concentrations of acetaldehyde. Although the amounts of undissociated hemoglobin following reaction with HMB did not increase with increasing concentrations of acetaldehyde, the significant increase of specific radioactivities of this fraction (152, 1967, and 6562 dpm/mg Hb for 0.1, 0.2, and 0.5 mM acetaldehyde, respectively) suggested possible crosslinks within the tetramer or dimer. The amino acid analysis of alpha- and beta-subunit adducts formed with 0.1 and 0.5 mM acetaldehyde showed that unreacted cysteine residues were more often detected at the higher acetaldehyde concentration consistent with the formation of cysteine adducts labile to acid hydrolysis or the shielding of cysteine residues in acetaldehyde-modified Hb against the subunit separation by HMB treatment. Thus acetaldehyde reacts differentially with the alpha- and beta-hemoglobin subunits and with the undissociated hemoglobin molecule.

Hydrogenation of a menhaden oil: I. Fatty acid and C20monoethylenic isomer compositions as a function of the degree of hydrogenation

AbstractUS menhaden oil is rich in long‐chain polyethylenic fatty acids, chiefly C20(eicosapentaenoic) and C22(docosahexaenoic) fatty acids, unlike Canadian herring oil, which is rich in long‐chain (C20and C22) monoethylenic fatty acids. An examination of the product fatty acids from hydrogenation of menhaden oil therefore comple‐ments studies previously published for herring oil. During a commer‐cial hydrogenation of menhaden oil, iodine value (IV) 159.0, on nickel catalyst, samples were collected at IV 150.0, 140.0, 131.5, 120.5, 96.5, 90.0 and 84.5. The fatty acid compositions were deter‐mined using a combination of mercuric adduct fractionation and gas liquid chromatographic (GLC) analyses, and the totaltranscontent by infrared spectroscopy. The partial hydrogenation resulted in the disappearance of the pentaenoic and hexaenoic fatty acids, a de‐crease in tetraenes, and a definite increase in trienes, 8.3% at IV 84.5 compared to 4.2% at IV 159.0. The dienoic fatty acids in‐creased to 13.2% at IV 84.5 compared to 4.1% at IV 159.0, and the monoenoic fatty acids increased to 34.2% from 24.0%. No impor‐tant changes in the saturated acids were observed, 43.8% at IV 84.5 compared to 41.6% at IV 159.0. The totaltranscontent varied from 3.4% at IV 150.0 to 45.1% at an IV of 84.5. The isomer composi‐tions of thecisandtransC20monoethylenic fatty acids were deter‐mined using a combination of preparative GLC, AgNO3thin layer chromatography and ozonolysis. Thecis20:1 acids at IV 84.5 still retained 27.5% of the major isomer (All) originally present at 72%. The parent A5, A8, All, A14 and A17 bonds of the 20:5 originally present could be detected in thecis20:1 isomers at IV 96.5 but not at IV 84.5. At IV 84.5, 58% of the 20:1was trans, the major isomer being All (9.4% of total 20.1), accompanied by important quanti‐ties of Δ10 and Δ12, respectively, 6.9% and 6.6% of the total 20:1.

Hydrogenation of a menhaden oil: II. Formation and evolution of the C20 dienoic and trienoic fatty acids as a function of the degree of hydrogenation

AbstractTo complement studies on monoethylenic fatty acids produced from the major polyunsaturated fatty acid (20:5A5,8,11,14,17) during hydrogenation of a menhaden oil of iodine value (IV) 159, the C10 dienoic and trienoic fatty acid isomers of partially hydro‐genated menhaden oils (PHMO) of IV 131.5, 96.5 and 85.5 were isolated by a combination of preparative gas liquid chromatography (GLC), mercuric adduct fractionation, and silver nitrate thin layer chromatography (AgNO3‐TLC). The 20:2 fatty acid methyl esters of the three PHMO samples were transformed to the corresponding alcohols and ozonized in BF3‐MeOH, followed by GLC analysis of the ozonolysis fragments. During the hydrogenation process, re‐sidual ethylenic bonds in the 20:2 isomers tend to migrate both towards the carboxyl group and towards the methyl end of the molecule. The hydrazine reaction results revealed that thetrans ethylenic bonds in the 20:2 and 20:3 isomers were distributed all along the the carbon chain, but thecis ethylenic bonds were more localized in the Δ11,Δ14 and Δ17 positions of the preexisting major menhaden oil component 20:5Δ5,8,11,14,17. Iatroscan analyses on AgNO3‐chromarods revealed that, as a result of the hydrogenation process, almost half of the 20:2 isomers were non‐methylene‐interruptedcis, trans/trans, cis structures.

Geochemistry of the thiourea adduct fraction from an Alberta petroleum

The thiourea adducted saturated hydrocarbon fraction (TUA) from a mature Alberta crude oil has been studied in detail. The predominating classes of compounds were found to be the acyclic isoprenoids, alkylcyclohexanes and alkylmethylcyclohexanes. Simulation experiments indicated that the alkylcyclohexanes and the alkylmethylcyclohexanes may have been formed by the advanced diagenesis of biologically occurring fatty acids.

Excision repair of DNA base damage in human cells treated with the chemical carcinogen 4-nitroquinoline 1-oxide

Excision repair of DNA damage produced by 4-nitroquinoline 1-oxide (4NQO), a potent chemical carcinogen, was compared in a normal human amnion FL cell line and a xeroderma pigmentosum (XP) cell line unable to repair ultraviolet-induced pyramidine dimers. The main objective of this study was to investigate, by a direct assay of the loss of damage from DNA, whether DNA damage induced by 4NQO in human cells is repaired by the excision-repair system as in Escherichia coli cells. DNA was extracted from FL and XP cells treated with [ 3H]4NQO, hydrolyzed and subjected to radiochromatographic analysis in order to quantitate the initial formation of 4NQO damage and subsequent disappearance during post-incubation. Two peaks of stable 4NQO-quanine adducts appeared on the chromatogram, together with one peak of stable 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide (4AQO) released from a labile fraction of 4NQO-guanine adduct during hydrolysis. The three kinds of stable 4NQO-purine adduct disappeared from DNA of the FL cells at almost the same rate of about 60% during 24-h post-incubation in culture medium, and 4AQO disappeared somewhat faster. In the XP cells, however, the stable adducts did not disappear from DNA, whereas about 40% of the 4AQO-releasing adduct disappeared from DNA. These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the XP cells are several times as sensitive as normal cells to killing by 4NQO. These results lead to the conclusion that in human cells 4NQO-induced lethality is mainly due to the four kinds of 4NQO-purine adduct as it is in E. coli, and that the adducts are excisable by the same excision-repair mechanism that works on pyramidine dimers.

The major cause of inactivation and mutation by 4-Nitroquinoline 1-Oxide in Escherichia coli: Excisable 4NQO-purine adducts

The potent carcinogen, 4-nitroquinoline 1-oxide, is known to mimic the biological effects of ultraviolet light on various living organisms. We conclude that the 4NQO † † Abbreviations used: 4NQO, 4-nitroquinoline 1-oxide; 4AQO, 4-aminoquinoline 1-oxide, 4HAQO, 4-hydroxyaminoquinaline-1-oxide. effects on Escherichia coli are mostly due to covalent binding of 4NQO to DNA producing 4NQO-guanine and 4NQO-adenine adducts in a ratio of about 4:1 without repair and about 7:1 after repair. This is based on the following experimental results. From E. coli cells treated with [ 3H]4NQO, DNA was extracted and subjected to radiochromatography. We detected two peaks of 4NQO-guanine adduct, one peak of 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide released from a labile fraction of 4NQO-guanine adducts during acid hydrolysis of DNA before streaking it on paper for chromatography. These four kinds of 4NQO-purine adducts disappeared from DNA of the normal strain at almost the same rate, about 85% in 60 minutes by post-incubating in nutrient broth, but these adducts did not disappear for the uvrA − derivative lacking the excision-repair ability for ultraviolet-induced pyrimidine dimers, except for slight disappearance of 4AQO-releasing adduct. The number of DNA lesions per genome of the uvrA − strain at 37% survival was found to be nearly equal between the 4NQO-purine adducts (~200 lesions) and pyrimidine dimers (~100 lesions). These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the uvrA − strain is about 25 to 30 times as sensitive as its parental strain to killing and mutation by either 4NQO or ultraviolet light. The unique characteristics of 4NQO-purine products are discussed in relation to the mutational specificity of 4NQO and the more-than-supposed complexity of excision repair for DNA.

Column chromatography of the bromo—mercuri—methoxy adducts of fatty acid methyl esters as a means of isolating polyenoic acids present in low concentrations

Due to the extreme complexity of oils such as animal tallows, determination of fatty acid composition by direct gas chromatographic examination of their unfractionated methyl esters is of limited utility. A more detailed analysis of animal fats has been achieved using a silicic acid column chromatographic method to resolve into classes of differing degree of unsaturation, the bromo—mercuri—methoxy adducts of the methyl esters. Methyl esters were recovered from the separated adduct fractions by treatment with acid and analysed by gas chromatography. This technique should be of general use in those cases where it is necessary to isolate small amounts of dienoic and polyenoic fatty acids from fats, as the required components are protected from autoxidative degradation during the isolation procedure.

Fatty acids of long chain length in baltic herring lipids

AbstractThe component fatty acids with carbon numbers exceeding 22 in flesh lipids of Baltic herring caught in May and September 1967 in the Turku archipelago have been studied. The total lipid content of the flesh of the herring was 3.5% on average in May and 7.2% on average in September. The fatty acids in the lipids were converted to methyl esters which were resolved and analyzed by urea adduct fractionation, thin layer chromatography and programmed temperature gas liquid chromatography (GLC). The lipids of the herring caught in May were found to contain 15 fatty acids with 24–32 carbon atoms, whereas the lipids of the herring caught in September were found to contain only nine fatty acids with 24–28 carbon atoms. The differences are probably due to nutritional factors. The long chain fatty acids in the lipids of the herring caught in September were isolated by preparative GLC and their structures were studied by UV spectroscopy before and after alkali isomerization, by IR spectroscopy and by GLC of their ozonization products. The identified acids were tetracosanoic, 15‐tetracosenoic, 12,15,18,21‐tetracosatetraenoic, 9,12,15,18,21‐tetracosapentaenoic, 6,9,12,15,18,21‐tetracosahexaenoic, 17‐hexacosenoic, 11,14,17,20,23‐hexacosapentaenoic, 8,11,14,17,20,23‐hexacosahexaenoic and 4,7,10,13,16,19,22‐octacosaheptaenoic acids. The proportion of the fatty acids containing over 22 carbon atoms in the lipids of fall herring is much higher than has been found earlier in the lipids of marine teleost fish; the reason may be due at least partly to differences in analytical methods.

Reaction of Methyl Linoleate with Mercuric Acetate in Acetic Acid

AbstractMethyl linoleate was reacted with mercuric acetate in acetic acid at 60°C for 2 hrs to give an oily mixture from which a relatively unstable adduct fraction with the composition close to C19H34O2(OCOCH3) (HgOCOCH3) was separated. The reaction of methyl linoleate with mercuric acetate at 115°C for 30 min. gave an oily mixture which was found to contain isomerized octadecadienoates, monoacetoxy and diacetoxy derivatives, and polymeric substances. It was found that the monoacetoxy derivatives contain 10‐acetoxy‐8,12‐, 12‐acetoxy‐9,13‐, 9‐acetoxy‐10,12‐ and 13‐acetoxy‐9,11‐octadecadienoates.

Composition of methyl esters from heat‐bodied linseed oils

AbstractTwo heat‐bodied linseed oils, with Gardner viscosities of 37 and 55 min, were saponified, converted to their methyl esters, and separated into 2 fractions with urea and methanol. Gas‐liquid chromatography showed the adduct fraction, which comprised 38舑41% of the total methyl esters, to contain: palmitic, stearic, oleic, 舠linoleic,舡 and trace amounts of 舠linolenic舡 acid. The nonadducting fraction (59舑62%) of the total methyl esters was separated by molecular distillation at 140C/7 ॖ into a distillate and residue. The distillate amounted to 18舑25% of the total methyl esters and had an iodine value (I.V.) of 142舑145; its absorption at 232 mॖ indicates 2.5舑3.0% conjugated diene. Hydrogenation of this distillate gave a liquid product with an iodine number of 4 and a pour point of 蜢50C. Gas chromatograms of the distillate and its hydrogenated derivative indicated at least 5舑7 components. Comparison of these peaks with known fatty acid methyl esters indicates that the components of these fractions were either cyclic or branched esters. The nonadducting residue fraction was composed mainly of polymeric acids.

Pressure reaction of maleic esters with vegetable oils

AbstractThe reaction of dimethyl maleate and some related dienophiles with vegetable oils under pressure has been studied successfully. When safflower oil reacts with 100% excess maleate, sulfur dioxide catalyst, and hydroquinone inhibitor at 290° for 1hr., 80 to 90% yields of adduct fraction, based on linoleate, are obtained. Under the same conditions almost equally good yields result with either linseed or soybean oils. With safflower oil, dimethyl fumarate gave slightly lower yields. Di‐n‐butyl maleate gave even poorer yields, and much residue was formed.

Reactions of dienophiles with vegetable oils. I. Reactions of maleic esters with sulfur dioxide catalyst

SummaryDirect reactions of dibutyl and dimethyl maleate with safflower oil were investigated. When transformed into esters and distilled, the products are generally different from the Diels‐Alder adducts obtained fromtrans,trans‐9,11‐linoleic acid. The differences most probably result from the formation of one or more isomers of the known product, of the substituted succinate, of a transannular lactone, or of a mixture of these and other isomers. No definite assignments of structure can yet be made. In one experiment the Diels‐Alder adduct was obtained as the major product from the reaction of dimethyl maleate with safflower oil.Reactions are best carried out by heating safflower oil to 290° while sweeping it with sulfur dioxide. The maleic ester is then added dropwise, and the product is transesterified and distilled. By this procedure a distillable adduct fraction is obtained in yields up to 70%, based on linoleate. Fumaric esters give similar results. Omission of the sulfur dioxide catalyst greatly reduces the yield.