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Ozone (O3) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O3 oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol...

Inhaled ground level ozone (O(3)) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O(3)-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O(3) reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O(3)-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O(3)-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O(3)-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O(3)-induced biological responses.

Inhalation of the ambient air pollutant ozone causes lung inflammation and can suppress host defense mechanisms, including impairing macrophage phagocytosis. Ozone reacts with cholesterol in the lung to form oxysterols, like secosterol A and secosterol B (SecoA and SecoB), which can form covalent adducts on cellular proteins. How oxysterol-protein adduction modifies the function of lung macrophages is unknown. Herein, we used a proteomic screen to identify lung macrophage proteins that form adducts with ozone-derived oxysterols. Functional ontology analysis of the adductome indicated that protein binding was a major function of adducted proteins. Further analysis of specific proteins forming adducts with SecoA identified the phagocytic receptors CD206 and CD64. Adduction of these receptors with ozone-derived oxysterols impaired ligand binding and corresponded with reduced macrophage phagocytosis. This work suggests a novel mechanism for the suppression of macrophage phagocytosis following ozone exposure through the generation of oxysterols and the formation of oxysterol-protein adducts on phagocytic receptors.

The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments., Graphical abstract fx1

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -β and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3. Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS.

Tyrosine-derived hydroperoxides are formed in peptides and proteins exposed to enzymatic or cellular sources of superoxide and oxidizing species as a result of the nearly diffusion-limited reaction between tyrosyl radical and superoxide. However, the structure of these products, which informs their reactivity in biology, has not been unequivocally established. We report here the complete characterization of the products formed in the addition of superoxide, generated from xanthine oxidase, to several peptide-derived tyrosyl radicals, formed from horseradish peroxidase. RP-HPLC, LC-MS, and NMR experiments indicate that the primary stable products of superoxide addition to tyrosyl radical are para-hydroperoxide derivatives (para relative to the position of the OH in tyrosine) that can be reduced to the corresponding para-alcohol. In the case of glycyl-tyrosine, a stable 3-(1-hydroperoxy-4-oxocyclohexa-2,5-dien-1-yl)-L-alanine was formed. In tyrosyl-glycine and Leu-enkephalin, which have N-terminal tyrosines, bicyclic indolic para-hydroperoxide derivatives were formed ((2S,3aR,7aR)-3a-hydroperoxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid) by the conjugate addition of the free amine to the cyclohexadienone. It was also found that significant amounts of the para-OH derivative were generated from the hydroxyl radical, formed on exposure of tyrosine-containing peptides to Fenton conditions. The para-OOH and para-OH derivatives are much more reactive than other tyrosine oxidation products and may play important roles in physiology and disease.

Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.

Dendrogenin A (DDA) is a tumor suppressor metabolite identified in human tissues that arises from the conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA) by a yet unidentified enzyme. DDA is present in the normal breast but its levels were found drastically decreased in breast tumors, showing that a deregulation of DDA metabolism occurred during breast carcinogenesis. It was shown that DDA displayed chemopreventive and anticancer properties (de Medina et al, Nat Commun, 2013; Voisin et al, PNAS, 2017; Segala et al, Nat Commun, in press). In addition, DDA blocks the biosynthesis of a newly identified cholesterol tumor promoter named 6-oxo-cholestan-3β,5α-diol (OCDO) (Voisin et al, PNAS, 2017). DDA and OCDO arise from 5,6-EC. We showed the existence of a metabolic balance between these two 5,6-EC derivatives in normal breast and BC that controls or stimulates BC progression (Silvente-Poirot & Poirot, Science, 2014, Voisin et al, PNAS, 2017). We addressed here the question of the identification and characterization of the DDA synthase (DDAS) and we determined whether its expression could reflect DDA levels in patient breast tumor and normal tissue. We report that the recombinant human glutathione transferase A1-1 (GST A1-1) produced DDA from 5,6α-EC and histamine (HA). The chemical characterization of the DDA product was performed by chromatography and mass spectrometry fragmentation. DDAS activity was found to be a new and important activity of GST A1-1 in addition to known glutathione transferase and steroid isomerase activities. The measured Michaelis constants of GST A1-1 for its new substrates were: Km5,6α-EC=0.27±0.05 µM and KmHA=0.35±0.3 µM, and the maximum velocity for the transformation of each substrates Vm5,6α-EC=0.81±0.2 µmol.min-1.mg and VmHA=0.66±0.2 µmol.min-1.mg. Interestingly, we showed that OCDO and other ring-B oxysterols, as well as several natural substrates and product of the GST A1-1, were potent inhibitors of DDAS activity while xenobiotics substrates of GST, and side chain oxysterols were not. Patient BC samples (n=50) showed significant decreased DDA levels and lower GST A1-1 protein expression compared to normal matched tissues, indicating that the decreased production of DDA in tumors is due to decreased expression of its enzyme. The analyses of two human BC mRNA databases from the Barts Cancer Institute (London, UK) and the Curie Institute (Paris, France) showed that the expression of GST A1-1 was lost in ER(+) BC tumors compared to normal breast tissue. Interestingly, DDAS was selectively expressed in the cytoplasm of epithelial cells from lactating ducts and lobular terminal units. Since these cells are the origin of most BC, the loss of DDAS expression and DDA biosynthesis combine to OCDO production, which controls DDAS activity, may constitute a major oncogenic process leading to BC development in human. Citation Format: Marc Poirot, Emmanuel Noguer, Florence Dalenc, Regis Soules, Lisa Barrett, Arnaud Rives, Hye-Young Kim, Brigitta Sjödin, Camille Franchet, Pilippe Rochaix, Raphaelle Duprez-Paumier, Magali Lacroix-Triki, Thomas Filleron, Leonor Chaltiel, Louise Jones, Emanuala Gadaleta, Claude Chalala, Sergio Roman-Roman, Thierry Dubois, Ned A. Porter, Bengt Mannervik, Michel Record, Sandrine Silvente-Poirot. Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5238.

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidycholine probe called PLPBSO (Tallmanet al.Chem. Res. Toxicol.2007, 7, 227−23417305406), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS−MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS−MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10(E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10(E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress., We describe the use of PLPBSO as a probe to identify major protein targets of oxidized phospholipids in human plasma. This approach identified apolipoprotein A1 (ApoA1) as the principal target of PLPBSO oxidation products and led us to map adducts from lipid oxidation products on this protein. We report the identification of adducts from several phospholipid electrophiles, including carboxy-terminal adducts. We further evaluated the sites of ApoA1 adduction by the prototypical lipid electrophile HNE. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress.

Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith–Lemli–Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway.

Oxidative stress gives rise to a number of electrophilic aldehydes from membrane phospholipids, and these compounds have been linked to pathophysiologic events associated with the progression of cardiovascular disease. A headgroup biotinylated phosphatidylcholine (PC) has been prepared, and its oxidation chemistry has been studied. Biotin or biotin-sulfoxide groups were attached to PC at the ammonium headgroup via a di-ethylene glycol link. The modified phospholipids have calorimetric and colloidal properties similar to those of the parent. The oxidation of PLPBSO (the biotin-sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidylcholine, PLPC) was studied under a variety of conditions. PLPBSO, like PLPC, undergoes oxidation to give electrophiles that adduct to small model peptides as well as to isolated proteins such as human serum albumin. PLPBSO incorporates into human blood plasma, and treatment of the plasma with water soluble free radical initiators gives rise to a number of biotinylated plasma proteins that can be isolated via (strept)avidin affinity. Isolated peptide or protein-lipid adducts can be identified by proteomics analyses, and studies on model peptides show that phospholipid-protein adduction sites can be identified by known algorithms. Biotinylated lipids such as PLPBSO and modern proteomics tools would appear to provide a new approach to exploring the chemistry and biology of membrane peroxidation associated with oxidative stress.

Fourth and fifth generation poly(propylene imine) dendrimers and methyl and benzyl functionalized copolymers of these dendrimers are solution blended with poly(vinyl chloride) (PVC). The methyl-derivative copolymer is observed to be dispersed in PVC as judged by optical and dynamic scanning calorimetry measurements. This dispersion leads to a substantial reduction in the glass transition temperature and a commensurate plasticization effect, demonstrating that functionalized dendrimer copolymers can successfully plasticize semicrystalline polymers. This plasticization is thought to occur as a result of additional free volume from the highly branched structure of the dendrimer.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants found in car exhausts, charbroiled food, and tobacco smoke. Three pathways for the metabolic activation of B[a]P to ultimate carcinogens have been proposed. The most widely accepted pathway involves cytochrome-P450 (CYP) 1A1- and/or 1B1-mediated formation of B[a]P-7,8-oxide, which undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further CYP1A1- and/or CYP1B1-mediated activation of the dihydrodiol results in the formation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. In previous studies, it was demonstrated that (+)-anti-B[a]PDE was the most potent tumorigen of the CYP-derived B[a]PDE diastereomers. We have developed a stable isotope dilution, liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay for all eight (±)-anti-B[a]PDE-derived dGuo and dAdo DNA-adducts. The LC-MRM/MS assay was rigorously validated and used to show that (+)-anti-trans-B[a]PDE-dGuo was the major adduct formed when naked DNA and human bronchoalveolar adenocarcinoma H358 cells were treated with (±)-anti-B[a]PDE. The preference for DNA-adducts derived from (+)-anti-B[a]PDE was even more apparent in cellular DNA. Thus, the increased potency of (+)-anti-B[a]PDE as a tumorigen is most likely due its ability to preferentially form DNA-adducts when compared with (−)-anti-B[a]PDE. Also, the adduct profile suggests that this occurs by binding of (+)-anti-B[a]PDE to DNA in a manner that facilitates covalent binding to dGuo rather than dAdo residues. Copyright © 2006 John Wiley & Sons, Ltd.

Poly(propyleneimine) (PPI) dendrimers and their end-functionalized derivatives are studied with steady and dynamic rheology to determine the influence of specific end groups. Fourth- and fifth- generation PPI dendrimers exhibit constant shear viscosities over a wide range of shear rates. Function- alizing the molecular end groups with methyl and benzyl acrylate monomers increases the glass transition temperature and leads to a significant elastic modulus and nonlinear rheological behavior at high shear rates. The zero-shear viscosity, corrected for the shift in glass transition, exhibits a unique maximum with molecular weight. The fractional free volume derived from the thermal rheological measurements is independent of the end-group derivatization and substantially larger than that for linear polymers.

The conformation of the trans-anti-(1S,2R,3S,4R)-N(2)-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in d(G(1)G(2)C(3)A(4)G(5)X(6)T(7)G(8)G(9)T(10)G(11)).d(C(12)A(13)C(14)C(15)A(16)C(17)C(18)T(19)G(20)C(21)C(22)), bearing codon 12 of the human N-ras protooncogene (underlined), was determined. This adduct had S stereochemistry at the benzylic carbon. Its occurrence in DNA is a consequence of trans opening by the deoxyguanosine amino group of (1R,2S,3S,4R)-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenyl-3,4-diol. The resonance frequencies, relative to the unmodified DNA, of the X(6) H1' and H6 protons were shifted downfield, whereas those of the C(18) and T(19) H1', H2', H2' ', and H3' deoxyribose protons were shifted upfield. The imino and amino resonances exhibited the expected sequential connectivities, suggesting no interruption of Watson-Crick pairing. A total of 426 interproton distances, including nine uniquely assigned BA-DNA distances, were used in the restrained molecular dynamics calculations. The refined structure showed that the benz[a]anthracene moiety bound in the minor groove, in the 5'-direction from the modified site. This was similar to the (+)-trans-anti-benzo[a]pyrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon [Cosman, M., De Los Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. It differed from the (-)-trans-anti-benzo[c]phenanthrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon, which intercalated in the 5'-direction [Lin, C. H., Huang, X., Kolbanovskii, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2001) J. Mol. Biol. 306, 1059-1080]. The results provided insight into how PAH molecular topology modulates adduct structure in duplex DNA.

Benzo[a]pyrene (1) can be converted to reactive electrophilic species by a number of metabolic pathways, of which the route to the mutagenic and carcinogenic diol epoxide(s) is the best studied. An alternative and interesting pathway to a highly genotoxic electrophile is through alkylation at the 6 position to 6-methylbenzo[a]pyrene (2) followed by oxidation of the methyl group to give 6-hydroxymethylbenzo[a]pyrene (3). Esterification of 3, especially to sulfate ester 4, gives compounds which are both mutagenic and carcinogenic. The major DNA adduct identified from exposure of rats and mice to 4 is the guanine N(2) adduct [2'-deoxy-N(2)-(benzo[a]pyren-6-ylmethyl)guanosine, 5] which is also formed via activation of 2 to a radical cation species by horseradish peroxidase/H(2)O(2) or iodine. To study the biological and structural properties of this adduct and the analogous adenine N(6) adduct (6), a nonbiomimetic synthesis of the adducted nucleosides 5 and 6 has been developed and has been extended to preparation of oligonucleotides containing 5 or 6 at a single site.

The mechanism of adduction of 2'-deoxyadenosine by styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides has been explored using (15)N(6)-labeled adenine nucleosides. The extent of reaction at N1 versus N(6) was evaluated by (1)H NMR of the N(6) adducts after allowing Dimroth rearrangement to occur. Products arising from attack at N1 followed by Dimroth rearrangement exhibited a small two-bond (1)H-(15)N coupling constant (N1-H2 J approximately 13 Hz); products from direct attack exhibited a much larger one-bond (1)H-(15)N coupling constant (J approximately 90 Hz). In the case of styrene oxide, all of the N(6) beta adduct arose by initial attack at N1, whereas the majority (70-80%) of the N(6) alpha adducts came from direct attack. The styrene oxide reaction was also studied with a self-complementary oligodeoxynucleotide (24-mer) containing nine (15)N(6)-labeled adenine residues. NMR examination of the N(6) alpha- and beta-styrene oxide adducts isolated after enzymatic degradation of the 24-mer gave very similar results, indicating that N1 attack can occur readily even with a duplexed oligonucleotide. With the PAH dihydrodiol epoxides, only naphthalene dihydrodiol epoxide exhibited significant initial reaction at N1 (50%). No detectable rearranged product was seen in reactions with benzo[a]pyrene dihydrodiol epoxide or non-bay or bay region benz[a]anthracene dihydrodiol epoxide; interestingly, a small amount of N1 attack (5-7%) was seen in the case of benzo[c]phenanthrene dihydrodiol epoxide. It appears that initial attack at N1 is only a significant reaction pathway for epoxides attached to a single aromatic ring.

The effect of adding acetylated poly(propyleneimine) dendrimers to the structure and rheology of aqueous solutions of high molecular weight poly(ethylene oxide) (PEO) was investigated by rheology and small-angle neutron scattering in a temperature range of 10–40 °C. In the semidilute regime, the steady shear rheology of PEO solutions was unmodified by the addition of dendrimers at a comparable weight concentration. At the highest concentrations studied, the addition of acetylated dendrimers suppressed the onset of a low-frequency elastic modulus at the lowest temperature investigated. For comparison, the addition of PEO of a comparable molecular weight at the same weight fraction resulted in a milder suppression but, unlike the dendrimers, greatly increased the solution viscosity. The addition of acetylated dendrimers to a semidilute PEO solution at 10 °C substantially reduced the solution turbidity. These effects on the rheology and optical properties were confirmed by small-angle neutron scattering measurements of the molecular structure of the mixture. Additional SANS measurements in the dilute regime (0.1 wt % PEO) showed quantitatively that the dendrimers decorated the PEO chains in a necklace structure, such as that observed previously for micelles. The results suggested a mechanism of rheology modification whereby the dendrimers disrupted the association network structure in the PEO solution at lower temperatures by preferentially associating with the PEO chains in solution. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 874–882, 2000

Commercially available fourth and fifth generation poly(propyleneimine) (PPI) dendrimers were functionalized with acetyl chloride and deuterated acetyl chloride. Their solution properties in water and D2O were measured with dilution viscometry, densitometry, rheology, and small-angle neutron scattering (SANS) and compared to molecular modeling. Both the acetylated and PPI dendrimers exhibited Newtonian rheology in solution at all concentrations, but the functionalized dendrimers were less viscous than the nonacetylated dendrimers at an equal weight fraction (50 wt %). The acetylated dendrimers exhibited a pronounced structure peak in SANS, however, that was not evident for PPI in solution and a greatly enhanced solubility. This structure peak, evident at concentrations as low as 0.2 wt %, was evidence for long-range electrostatic interdendrimer forces, which were screened by added salt. A quantitative agreement was obtained between the dilute-limiting absolute scattering spectra of both the nonacetylated and acetylated dendrimers in solution with model calculations via a homogeneous spherical model and input parameters independently obtained from dilution viscometry or direct calculation. The combined measurements verified significant solvent penetration for both dendrimer types. The form factors measured in this manner were also in good quantitative agreement with the results of molecular dynamics simulations, which pointed to significant backfolding of the terminal groups. SANS and rheology measurements at higher concentrations suggested dendrimer clustering and interpenetration with increasing concentration, leading to less structure and lower viscosity than would be predicted from the dilute-limiting behavior. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 857–873, 2000

The structure of the non-bay region (8S,9R,10S,11R)-N(6)-[11-(8,9,10, 11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2'-de oxyadenosyl adduct at X(6) of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined. Molecular dynamics simulations were restrained by 475 NOEs from (1)H NMR. The benz[a]anthracene moiety intercalated above the 5'-face of the modified base pair and from the major groove. The duplex suffered distortion at and immediately adjacent to the adduct site. This was evidenced by the disruption of the Watson-Crick base pairing for X(6) x T(17) and A(7) x T(16) and the increased rise of 7.7 A between base pairs C(5) x G(18) and X(6) x T(17). Increased disorder was observed as excess line width of proton resonances near the lesion site. Comparison with the bay region benzo[a]pyrene [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224] and bay region benz[a]anthracene [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981] adducts with the corresponding stereochemistry and at the same site shows that this non-bay region benz[a]anthracene lesion assumes different base pair geometry, in addition to exhibiting greater disorder. These differences are attributed to the loss of the bay region ring. The results suggest the bay region ring contributes to base stacking interactions at the lesion site. These structural differences between the non-bay and bay region lesions are correlated with site-specific mutagenesis data. The bay region benzo[a]pyrene and bay region benz[a]anthracene adducts were poorly replicated in vivo, and induced A --> G mutations. In contrast, the non-bay region benz[a]anthracene adduct was easily bypassed in vivo and was nonmutagenic.

Tetrabutylammonium triphenydifluorosilicate (TBAT) has been found to be a useful reagent for the conversion of 6-chloropurine nucleosides to 6-fluoropurine derivatives. The 6-chloropurine nucleosides were reacted with trimethylamine to form quaternary trimethylammonium salts which were treated in situ with TBAT in DMF to effect conversion to the 6-fluoro derivatives in yields of 59–72%.

Fully deprotected oligodeoxynucleotides containing 6-chloropurine have been synthesized and used in solution phase reactions with amine nucleophiles to prepare oligonucleotides containing substituted adenine residues. This strategy was used for the preparation of a double-stranded oligonucleotide crosslinked by a 4-carbon tether between N 6 positions of deoxyadenosines in the two strands.

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.

Cholesterol is synthesized endogenously in the brain in a complex pathway that starts with the condensation of acetate and acetoacetate. The last step in this pathway is the conversion of 7-dehydrocholesterol (7-DHC) into cholesterol, a step catalyzed by the enzyme dehydrocholesterol reductase 7 (DHCR7). Mutations in this enzyme lead to the accumulation of 7-DHC, a hallmark of the disorder known as Smith-Lemli-Opitz Syndrome (SLOS). The sterol profile in SLOS cells is characterized by lower levels of desmosterol and cholesterol and higher than normal levels of 7-DHC, the most oxidizable lipid known to date. As a tentative approach to improve this phenotype, we tested in this study the effects of ketone bodies using well-established cell culture models of SLOS. WT and Dhcr7-deficient Neuro2a cells were supplemented with different concentrations of acetoacetate (AcAc) and β-hydroxybutyrate and the sterol profile was analyzed. The levels of five sterols in the post-lanosterol pathway, including 7-DHC and cholesterol, were quantified using UHPLC-MS/MS and several isotopically labeled internal standards. Supplementing Dhcr7-deficient Neuro2a cells with AcAc for 96 hours led to a decrease in 7-DHC and an increase in desmosterol and cholesterol when compared to the control conditions, suggesting an improvement in the sterol profile. On the other hand, the same treatment in WT Neuro2a cells did not affect 7-DHC levels, indicating that the ketone bodies – especially AcAc – could have beneficial effects in SLOS cells. These observations are now being confirmed in primary human fibroblasts taken from healthy and SLOS patients. In conclusion, these results suggest that ketone bodies can affect positively the cholesterol biosynthesis in cultured SLOS cells, where they improve the sterol profile. Support NIH, NICHD.

Rat sperm tail fibrous sheath was isolated using mechanical and chemical dissection methods from spermatozoa collected from the cauda epididymis. The procedures used to isolate the fibrous sheath were monitored by phase-contrast microscopy and purity was verified by electron microscopy. SDS-PAGE of isolated total fibrous sheath revealed at least 17 bands when stained with Coomassie brilliant blue and 20 bands with silver stain. The most intensely staining proteins, using both staining methods, were a double band at 80-87 kDa, and a band at 28.5 kDa, whereas with silver staining, bands at 66.2 kDa and 32.7 kDa were also intensely stained. Electroelution following SDS-PAGE was used to isolate II of these proteins (l16.4, 87.5, 80.9, 66.2, 57.2, 49.7, 46.8, 37.3, 32.7, 28.5 and 15.5 kDa). Amino acid analysis revealed that these proteins were abundant in aspartic and glutamic acid, glycine, serine and leucine, while histidine and phenylalanine were of low abundance. The content of cystine varied widely from 9.4% to 1.4%. The amino termini of the 80.9 kDa, 32.7 kDa, 28.5 kDa and 15.5 kDa proteins were blocked. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat fibrous sheath was localized to the principal piece of the rat, rabbit and human spermatozoa, but in the rabbit it also labelled the equatorial region of the head. Western blotting detected all protein bands in isolated fibrous sheath and a similar range of proteins in the spermatozoa of rat and rabbit. Human sperm proteins of 116 kDa and 80 kDa were immunoreactive in common with other species, and there was only weak crossreactivity with the other proteins. These data suggest the presence of common epitopes in the proteins of all three species.

Oligonucleotides bearing adducts of the diol epoxides of benzo[a]pyrene and other polycyclic aromatic hydrocarbons on the exocyclic amino group of adenine have been prepared in a regio and stereospecific manner. The strategy involves reversal of the electrophile-nucleophile relationship; the PAH is linked to the adenine nucleus via condensation of an aminotriol (derived from the diol epoxide) with 6−fluoropurine deoxyribonucleoside. This condensation is carried out after the fluoro nucleoside has been incorporated into the oligonucleotide, but while the oligomer is still immobilized on the solid matrix. The diastereomeric oligomers derived from (±)−anti−BPDE are readily separable by HPLC, thus circumventing the need for individual enantiomers of the diol epoxide.

Dimensionless equilibrium constants describing the self-association of the hexafluoro-2-alkyl-2-propanol group have been determined from infrared spectroscopic data. Corresponding values of these equilibrium constants for a fully modified polyisoprene containing the hexafluoroisopropanol group (PHFPI) were calculated by taking into account differences in the molar volume of the model and the specific repeat unit of the polymer. Equilibrium constants describing the inter-association of PHFPI with methacrylate, acrylate, and acetoxy type carbonyl groups were obtained from spectroscopic studies of miscible PHFPI blends with poly(n-butyl methacrylate), poly(methyl acrylate), and an ethylene-co-vinyl acetate copolymer containing 70 wt% vinyl acetate. The set of equilibrium constant values were then used to calculate theoretical miscibility windows for the complete range of PHFPI blends with poly(n-alkyl methacrylate)s and four copolymers, ethylene-co-methyl methacrylate, styrene-co-methyl acrylate, ethylene-co-methyl acrylate, and ethylene-co-vinyl acetate. Experimental infrared studies confirm the general validity of the predicted miscibility windows. © 1994 John Wiley & Sons, Inc.

Polyisoprene (PIP) was found to react with trifluoroacetic acid (TFA) to give an adduct. Saponification of the ester gave a new alternating copolymer of ethylene and α-methyl vinyl alcohol. TFA did not react with polybutadiene (PBD) under these conditions, thus providing a way to produce amphiphilic block copolymers from PBD-b-PIP. TFA addition to the PIP block took place cleanly at an ambient temperature with 2 equiv of the acid in toluene to give block copolymer of PBD and trifluoroacetated PIP. This polymer is very soluble in toluene regardless of molecular weight. Methanolysis with NaOMe cleaved the ester to give PDB-b-(ethylene-alt-α-methyl vinyl alcohol) (PIPOH) in a MeOH/toluene mixture

Highly branched polyphenylenes, polyphenylene ethers, aromatic polyesters and polyamides synthesized from AB2 type monomers are reviewed. Polyphenylenes were obtained by aryl-aryl coupling reactions of 3,5-dihalo-phenyl organometallic reagents. 13C NMR indicates about 70% branching efficiency. A Tgat 236°C was observed, but the polymer did not form films. Polyethers were prepared by oxidative coupling of 2,4,6-tribromophenol and 2,4-dibromophenol. The former monomer gave high molecular weight polymers but the latter monomer did not polymerize well. Condensation of 3,5-diaminobenzoic acid (I) and 3-aminoisophthalic acid derivatives in an amide solvent gave lyotropic hyperbranched aromatic polyamides. The GPC indicated a large degree of polymer aggregation in the amide solvent in the absence of a complexing salt.

The formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be post labeled with azidobiotin tags by Cu+-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.

Tetrabutylammonium triphenydifluorosilicate (TBAT) has been found to be a useful reagent for the conversion of 6-chloropurine nucleosides to 6-fluoropurine derivatives. The 6-chloropurine nucleosides were reacted with trimethylamine to form quaternary trimethylammonium salts which were treated in situ with TBAT in DMF to effect conversion to the 6-fluoro derivatives in yields of 59–72%.

Acrolein reacts with dG to form hydroxylated 1,N(2)-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2a] purin-10(3H)-ones, commonly referred to as the gamma-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the N(2)-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence. Here we report the structure of the stereochemically favored (R)-gamma-hydroxytrimethylene N(2)-dG:N(2)-dG interstrand DNA cross-link in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3' x 5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' (X(7) is the dG linked to the alpha-carbon of the carbinolamine linkage, and Y(19) is the dG linked to the gamma-carbon of the carbinolamine linkage; the cross-link is in the 5'-CpG-3' sequence). The structure was characterized using isotope-edited (15)N nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X(7) or Y(19) were (15)N-labeled. Analyses of NOE intensities involving Y(19) N(2)H indicated that the (R)-gamma-hydroxytrimethylene linkage was the major cross-link species, constituting 80-90% of the cross-link. The X(7) and Y(19) imino resonances were observed at 65 degrees C. Additionally, for the 5'-neighbor base pair G(5) x C(20), the G(5) imino resonance remained sharp at 55 degrees C but broadened at 65 degrees C. In contrast, for the 3'-neighbor A(8) x T(17) base pair, the T(17) imino resonance was severely broadened at 55 degrees C. Structural refinement using NOE distance restraints obtained from isotope-edited (15)N NOESY-HSQC data indicated that the (R)-gamma-hydroxytrimethylene linkage maintained the C(6) x Y(19) and X(7) x C(18) base pairs with minimal structural perturbations. The (R)-gamma-hydroxytrimethylene linkage was located in the minor groove. The X(7) N(2) and Y(19) N(2) atoms were in the gauche conformation with respect to the linkage, which maintained Watson-Crick hydrogen bonding of the cross-linked base pairs. The anti conformation of the hydroxyl group with respect to C(alpha) of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C(20) O(2) located in the 5'-neighboring base pair G(5) x C(20). The formation of this hydrogen bond may, in part, explain the thermal stability of this carbinolamine interstrand cross-link and the stereochemical preference for the (R) configuration of the cross-link.

HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.

Polyisoprene reacts with halogenated acetic acids such as trifluoro-, trichloro- and dichloroacetic acid to give carboxy polyalkanes. The reactivities of trans- and cis-polyisoprene are similar. Saponification of the modified polymer with NaoCH 3 gives e tert-hydroxyl-containing polyalkane, namely an alternating copolymer of ethylene and α-methylvinyl alcohol. This polymer slowly hardens at room temperature to give a plastic material with a T g at 45-70°C. Its tensile mechanical properties exhibit low elongation of break and stiffness.

Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. They yield interstrand DNA cross-links between guanines in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, as a result of opening of the 1,N(2)-gamma-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, that maintain the Watson-Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N(2)-gamma-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing.

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.

Because alpha-tocopherol (alpha-TOH) mediates the peroxidation of cholesterol-esterified lipids in human low-density lipoprotein (LDL) in vitro and has displayed disappointing results against the onset and development of atherosclerosis, it may not be appropriate for use as a therapeutic in the prevention and/or treatment of the disease. Herein are described the experimental results demonstrating that 3-pyridinols spare alpha-TOH, do not efficiently mediate lipid peroxidation, and protect lipoprotein tryptophan residues in human LDL.

The solution structure of the (-)-(1R,2S,3R,4S)-N6-[1-(1,2,3, 4-tetrahydroxy-benz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined. This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR. The precision of the refined structures was monitored by pairwise root-mean-square deviations which were1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms. The refined structure was a right-handed duplex, in which the benz[a]anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17. In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17. These two base pairs also buckled in opposite directions away from the intercalated BA moiety. The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10)-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224]. Both adducts intercalated toward the 5'-direction from the site of adduction. The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.

Polycyclic aromatic hydrocarbons are metabolized to a wide variety of oxidized derivatives, including highly reactive diol epoxides which alkylate DNA. The reaction lacks regio- or stereospecificity but occurs primarily at the exocyclic amino groups of deoxyguanosine and deoxyadenosine. An efficient route to N 6 adducts of deoxyadenosine is described using as examples those arising from trans opening of the anti -tetrahydrodiol epoxides of naphthalene, benzo[ a ]pyrene, and benzo[ c ]phenanthrene. The adducts were synthesized in 50–92% yields by reaction of 6-fluoropurine 2′-deoxyriboside with aminotriols formed by trans opening of racemic dihydrodiol epoxides using liquid NH 3 . The diastereomeric adducts were separated by HPLC and their absolute configurations were assigned by circular dichroism. 1 H NMR studies revealed significant differences in conformation of the tetrahydroaromatic ring between the sterically unrestricted naphthalene derivative and the sterically congested derivatives of benzo[ a ]pyrene and benzo[ c ]phenanthrene. These differences may have a bearing on the higher carcinogenicity shown by the latter hydrocarbons. Undecadeoxyoligonucleotides bearing regio- and stereochemically defined adenine N 6 - anti - trans -benzo[ a ]pyrene adducts have been prepared.

Spectroscopic evidence is presented for the formation of a carbinolamine interchain cross-link in 5'-CpG-3' sequences, arising from the acrolein adduct gamma-OH-PdG. This may be important in understanding biological processing of acrolein-induced DNA damage in CpG sequences.

Rat outer dense fibres were isolated from cauda epididymal spermatozoa using mechanical and chemical dissection methods. Sperm tail isolation procedures were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated outer dense fibres revealed at least nine Coomassie brilliant blue stained bands, and 12 silver staining bands. The most abundant proteins were a large band between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolated proteins showed similar compositions, being abundant in aspartic and glutamic acid, serine, glycine and leucine. However, the content of cysteine and proline was highly variable among the isolated proteins. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat outer dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum also showed crossreactivity in the perforatorium of rat sperm heads and the acrosome and equatorial segment of rabbit sperm heads. These data indicate that it is technically possible to isolate proteins from the outer dense fibres that will enable further studies of the amino acid sequences of sperm tail proteins.