Production Of Adducts Research Articles

Alkali Metal Cation-Induced Destabilization of Gas-Phase Protein–Ligand Complexes: Consequences and Prevention

Electrospray ionization, now a well established technique for studying noncovalent protein-ligand interactions, is prone to production of alkali metal adducts. Here it is shown that this adduction significantly destabilizes the interactions between two model proteins and their ligands and that destabilization correlates with cation size. For both the [FKBP·FK506] and [lysozyme·NAG(n)] systems, dissociation of the metalated complex occurs at markedly lower collision energies than their purely protonated equivalents. Dependency upon size of the metal(+) demonstrates the importance of electrostatic charge density during the dissociation process. Differences in the gas phase basicities (GBapp) of the multiply charged protein ions and proton and sodium affinities of the ligands explain the observed charge partitioning during dissociation of the complexes. Ion mobility-mass spectrometry measurements demonstrate that metal cation adduction does not induce a significant increase in unfolding of the polypeptides, indicating that this is not the principal mechanism responsible for destabilization. Destabilizing effects can be largely reduced by exposing the electrospray to solvent (e.g., acetonitrile) vapor, a method that acts to reduce the amount of adduct formation as well as decrease the charge states of the resulting ions. This approach leads to more accurate determination of apparent K(D)s in the presence of trace alkali metals.

Raman Spectroscopy of the Reaction of Thin Films of Solid-State Benzene with Vapor-Deposited Ag, Mg, and Al

Thin films of solid-state benzene at 30 K were reacted with small quantities of vapor-deposited Ag, Mg, and Al under ultrahigh vacuum, and products were monitored using surface Raman spectroscopy. Although Ag and Mg produce small amounts of metal–benzene adduct products, the resulting Raman spectra are dominated by surface enhancement of the normal benzene modes from metallic nanoparticles suggesting rapid Ag or Mg metallization of the film. In contrast, large quantities of Al adduct products are observed. Vibrational modes of the products in all three systems suggest adducts that are formed through a pathway initiated by an electron transfer reaction. The difference in reactivity between these metals is ascribed to differences in ionization potential of the metal atoms; ionization potential values for Ag and Mg are similar but larger than that for Al. These studies demonstrate the importance of atomic parameters, such as ionization potential, in solid-state metal–organic reaction chemistry.

Biologiczne skutki stresu oksydacyjnego wywołanego działaniem pestycydów

Pesticides are used to protect plants and numerous plant products. They are also utilized in several industrial branches. These compounds are highly toxic to living organisms. In spite of close supervision in the use of pesticides there is a serious risk that these agents are able to spread into the environment and contaminate water, soil, food, and feedstuffs. Recently, more and more studies have been focused on understanding the toxic mechanisms of pesticide actions. The data indicate that the toxic action of pesticides may include the induction of oxidative stress and accumulation of free radicals in the cell. Long-lasting or acute oxidative stress disturbs cell metabolism and is able to produce permanent changes in the structure of proteins, lipids, and DNA. The proteins that are oxidized may lose or enhance their activity. Moreover, the proteins oxidized are able to form aggregates that inhibit the systems responsible for protein degradation and lead to alterations of proteins in the cell. Once oxidized, lipids have the capacity to damage and depolarize cytoplasmic membranes. Free oxygen radicals are harmful to DNA including damage to single nitric bases, DNA strand breaks and adduct production. Many studies indicate that oxidative stress may accelerate development of numerous diseases including cancer and neurodegenerative ones such as Alzheimer’s and Parkinson’s disease and may also be responsible for infertility.

Physiological and Molecular Characterization of Aristolochic Acid Transport by the Kidney

Consumption of herbal medicines derived from Aristolochia plants is associated with a progressive tubulointerstitial disease known as aristolochic acid (AA) nephropathy. The nephrotoxin produced naturally by these plants is AA-I, a nitrophenanthrene carboxylic acid that selectively targets the proximal tubule. This nephron segment is prone to toxic injury because of its role in secretory elimination of drugs and other xenobiotics. Here, we characterize the handling of AA-I by membrane transporters involved in renal organic anion transport. Uptake assays in heterologous expression systems identified murine organic anion transporters (mOat1, mOat2, and mOat3) as capable of mediating transport of AA-I. Kinetic analyses showed that all three transporters have an affinity for AA-I in the submicromolar range and thus are likely to operate at toxicologically relevant concentrations in vivo. Structure-activity relationships revealed that the carboxyl group is critical for high-affinity interaction of AA-I with mOat1, mOat2, and mOat3, whereas the nitro group is required only by mOat1. Furthermore, the 8-methoxy group, although essential for toxicity, was not requisite for transport. Mouse renal cortical slices avidly accumulated AA-I, achieving slice-to-medium concentration ratios >10. Uptake by slices was sensitive to known mOat1 and mOat3 substrates and the organic anion transport inhibitor probenecid, which also blocked the production of DNA adducts formed with reactive intracellular metabolites of AA-I. Taken together, these findings indicate that OAT family members mediate high-affinity transport of AA-I and may be involved in the site-selective toxicity and renal elimination of this nephrotoxin.

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Dopa decarboxylase (DDC or AADC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-aromatic amino acids into the corresponding aromatic amines. AADC deficiency is an inborn error of neurotransmitters biosynthesis with an autosomal recessive inheritance. About 30 pathogenic mutations have been identified, but the enzymatic phenotypes causing AADC deficiency are unknown, and the therapeutic management is challenging. Here, we report biochemical and bioinformatic analyses of the human wild-type DDC and the pathogenic variants G102S, F309L, S147R and A275T whose mutations concern amino acid residues at or near the active site. We found that the mutations cause, even if to different extents, a decreased PLP binding affinity (in the range 1.4-170-fold), an altered state of the bound coenzyme and of its microenvironment, and a reduced catalytic efficiency (in the range 17-930-fold). Moreover, as compared to wild-type, the external aldimines formed by the variants with L-aromatic amino acids exhibit different spectroscopic features, do not protect against limited proteolysis, and lead to the formation, in addition to aromatic amines, of cyclic-substrate adducts. This suggests that these external Schiff bases are not properly oriented and anchored, i.e., in a conformation not completely productive for decarboxylation. The external aldimines that the variants form with D-Dopa also appear not to be correctly located at their active site, as suggested by the rate constants of PLP-L-Dopa adduct production higher than that of the wild-type. The possible therapeutic implications of the data are discussed in the light of the molecular defects of the pathogenic variants.

Characterization of estrogen quinone-derived protein adducts and their identification in human serum albumin derived from breast cancer patients and healthy controls

Both 17β-estradiol-2,3-quinone (E 2-2,3-Q) and 17β-estradiol-3,4-quinone (E 2-3,4-Q) are reactive metabolites of estrogen that are thought to be responsible for the estrogen-induced genotoxicity. The aim of this study was to establish a methodology to analyze estrogen quinone-derived protein adducts and to measure the background levels of these adducts in human serum albumin (Alb) derived from female blood donors in Taiwan. Results from in vitro experiments confirmed that the production of estrogen quinone-derived adducts on serum Alb increased with increased concentration of estrogen quinones. Time-course experiments suggested that both E 2-2,3-Q- and E 2-3,4-Q-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter for up to 24 h. Additionally, with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) pretreatment, the production of estrogen quinone-derived protein adducts was detected in human MCF-7 breast cancer cells exposed to estrogen. Co-treatment of a catechol-O-methyl transferase inhibitor further enhanced the production of estrogen quinone-derived adducts in all cases. When we investigated the levels of estrogen quinone-derived adducts in human serum Alb, cysteinyl adducts of E 2-2,3-Q-1-S-Alb, E 2-2,3-Q-4-S-Alb, and E 2-3,4-Q-2-S-Alb were detected in all healthy female controls ( n = 10) with median levels at 147 (range 14.1–533), 197 (range 30.0–777), and 65.6 (range 17.6–1360) (pmol/g), respectively. We noticed that levels of E 2-2,3-Q-derived adducts were 2-fold greater than those of E 2-3,4-Q-2-S-Alb in controls whereas levels of E 2-3,4-Q-2-S-Alb were 2-fold higher than those of E 2-2,3-Q-derived adducts in patients ( n = 20). Additionally, levels of E 2-2,3-Q-4-S-Alb correlated significantly with those of E 2-3,4-Q-2-S-Alb (correlation coefficient r = 0.684–0.850, p < 0.05). Overall, we conclude that cumulative body burden of E 2-3,4-Q is a significant predictor of breast cancer.

Characterization of Intermediates in the C−F Activation of Tetrafluorobenzenes using a Reactive Ni(PEt3)2Synthon: Combined Computational and Experimental Investigation

The reaction of the anthracene adduct (PEt3)2Ni(η2-C14H10) with 1,2,3,4-tetrafluorobenzene revealed NMR spectroscopic evidence for the immediate equilibrium production of the dinuclear adduct [(PEt3)2Ni]2(μ-η2:η2-C6F4H2) and mononuclear adducts (PEt3)2Ni(η2-C6F4H2). Similarly, the reaction of (PEt3)2Ni(η2-C14H10) with 1,2,4,5- and 1,2,3,5-tetrafluorobenzene revealed NMR spectroscopic evidence for the equilibrium production of the dinuclear adducts and mononuclear adducts and also the C−H activation products (PEt3)2NiH(C6F4H). The conversions to both the adducts and C−H activation products were too minimal to allow for isolation, and the low concentrations hampered complete NMR spectroscopic characterization. The reaction of the readily prepared (PEt3)2NiMe2 with isobutene allowed for the in situ generation of (PEt3)2Ni(η2-H2C═CMe2) as a more reactive Ni(PEt3)2 synthon. The reaction of (PEt3)2Ni(η2-H2C═CMe2) with tetrafluorobenzenes also generates small equilibrium concentrations of adducts and C−H activation products, but the equilibrium can be driven by the removal of isobutene. The dinuclear adducts [(PEt3)2Ni]2(μ-η2:η2-1,2,3,4-C6F4H2) and [(PEt3)2Ni]2(μ-η2:η2-1,2,4,5-C6F4H2) were isolated and fully characterized by X-ray crystallography. The isolation of the dinuclear adduct of 1,2,3,5-tetrafluorobenzene was hampered by its rapid C−F activation and the generation of the less soluble aryne adduct [(PEt3)2Ni]2(μ-η2:η2-3,4,6-C6F3H), which was fully characterized. The spectroscopic characterization of all the tetrafluorobenzene mononuclear and dinuclear adducts as well as C−H activation products was accomplished by variable-temperature NMR spectroscopy and aided by DFT calculations on the proposed intermediates. The observation of equilibrium concentrations of the intermediates in solution allowed for the determination of the thermodynamic parameters for their interconversion.

Synthesis of Chlorinated Bicyclic Adduct as Biocids for Sulfate-Reducing Bacteria

Synthesis of bicyclic systems containing chlorine atoms, and/or ether groups in aromatic rings can be con- sidered as an important method for building bicyclic system and production of new adducts. One of the most important types in the cycloaddition reaction is the Diels-Alder reaction (1,4 cycloaddition). In the present investigation a new ether of allylic type (dienophile) p-allyl bromo phenol was prepared and its structure was confirmed by molecular weight determination, refractive index, infrared spectra, and density. A new adduct was obtained by means of 1,4 cycloaddition reaction of hexachlorocyclopentadiene (HCP) and the new pre- pared dienophile. The reaction takes place without using solvent, catalysts, or elimination of any compound. The effect of variations in temperature, initial molar ratio and reaction duration were studied to determine the optimum conditions of the reaction. The optimum conditions reached were reaction temperature recorded 140?C, initial molar ratio diene: dienophile was 3:1 and the reaction duration time reached 6 h. Under these optimum conditions the maximum yield was 78%. The new adduct revealed very high biological effect as sulfate-reducing bacteria (SRB).

Human mesangial cells resist glycoxidative stress through an antioxidant response

The generation of advanced glycation end-products (AGE), the interaction with their receptors, the generation of reactive oxygen species, and the modulation of intracellular redox equilibrium are believed to be the main factors causing alterations of mesangial cell physiology leading to diabetic nephropathy. Normal human primary mesangial cells were exposed to glycoxidative stress by culture in high glucose (HG) or treatment with AGE for up to 6 days. In both cases only a moderate generation of reactive oxygen species and production of HNE-protein adducts were induced while protein nitrotyrosination was not affected. Moreover, HG and AGE caused a significant antioxidant response, confirmed by the induction of heme oxygenase 1 and the consumption of vitamin E. Glutathione was decreased only by HG. Mesangial cell proliferation and viability were slightly affected by HG and AGE. Furthermore, both treatments failed to influence TGF-ß1 and MCP-1 secretion and to modulate RAGE and collagen IV expression. We believe that normal human mesangial cells can resist glycoxidative stress by the observed antioxidant response. These results support the concept that mesangial cells are only partly responsible for the onset and progression of diabetic nephropathy and that the role of other cell types, such as podocytes and endothelial cells, should be taken into consideration.

The room temperature chemistries of isocyanates with zeolite NaX

Isocyanates and di-isocyanates are used in the production of various chemicals and materials, such as pesticides and polyurethane. However, since the disastrous accidental release of isocyanate in Bhopal in 1984 this class of toxic compounds has been under scrutiny, with growing interest in means to respond to isocyanate release. The susceptibility of isocyanates to nucleophilic substitution [D.P.N. Satchell, R.S. Satchell, Chem. Soc. Rev. 4 (1975) 231] and the known nucleophilicity of NaX [Yang, et al., Microporous Mesoporous Mater. 92 (2006) 56; Doetschman, et al., Microporous Mesoporous Mater. 92 (2006) 292; Kanyi, et al., Microporous Mesoporous Mater. 108 (2008) 103] led to this study of a spectrum of isocyanates representing cyclic and aromatic isocyanates, di-isocyanates, 1°, 2°, and 3° isocyanates, and alkyl chain isocyanates of various lengths. Additional experiments were done in order to understand the nature of a tightly bound chemical adduct product that forms in the isocyanate reactions with the zeolite. The isocyanate functional group was shown to be susceptible to nucleophilic attack by Faujasite NaX. A carbamate framework adduct, with the anionic nitrogen stabilized by the sodium cation is proposed. The product is very tightly bound to the zeolite. In two, but not all, isocyanates in which there are methyl protons β to the isocyanate moiety, an elimination reaction took place to form the α, β olefin, competing with the nucleophilic addition reaction. The types of chemistry occurring and the tenacity with which the products are retained, suggests that zeolites may have practical applications in hazardous materials cleanup or controlling isocyanate vapor release.

RCM-based approach to seven- and eight-member ring-fused β-carbolines

RCM-based new route to the synthesis of seven- and eight-member ring-fused β-carboline from Morita–Baylis–Hillman adduct or Barbier-allylation product of N-allyl-1-formyl-9 H-β-carboline is described.

Comparative EPR study of different macrophage types stimulated for superoxide and nitric oxide production

Despite the major impact of ROS on human health, their quantification remains difficult and requires an analytical approach, such as the EPR spin trap technique. In this study, a comparative EPR analysis of different macrophage types stimulated for superoxide and nitric oxide production was performed. U937 monocytes, J774A.1, RAW 264.7 and primary mouse (PMM) macrophages were included. In contrast to the U937 cells, all macrophages produced significant EPR signals after stimulation. The use of PMA as stimulator and CM-H as spin probe led to the highest response in EPR signals for detection of O2·− as nitroxide radical. A combination of LPS and IFN-gamma and the spin trap [Fe(DETC)2] turned out to be the best combination for the production and detection of intracellular NO spin adducts. In conclusion, this study established practical experimental conditions for the EPR analysis of O2·− and NO produced by different types of activated macrophages.

Abstract 4712: Characterization of estrogen quinone-derived protein adducts and their identification in human serum albumin

Abstract Both estrogen-2,3-quinone (E2-2,3-Q) and estrogen-3,4-quinone (E2-3,4-Q) are reactive metabolites of estrogen that are thought to be responsible for the estrogen-induced genotoxicity. The aim of this study was to establish a methodology to analyze estrogen quinone-derived protein adducts and to measure the background levels of these adducts in human serum albumin (Alb) derived from female blood donors in Taiwan. The methodology, which employs trifluoroacetic acid anhydride and methanesulfonic acid to selectively cleave cysteinyl adducts, had previously been applied to measure naphthalene adducts in blood proteins. Results from in vitro experiments confirmed that the production of estrogen quinone-derived adducts on serum albumin increased with increased concentration of estrogen quinones (0-100 nM). The average formation of estrogen quinone-derived cysteinyl adducts per unit concentration of estrogen quinones was estimated to be 13.0, 48.2, and 36.1 (pmol/g Alb)/(nM) for E2-2,3-Q-1-S-Alb, E2-2,3-Q-4-S-Alb, and E2-3,4-Q-2-S-Alb, respectively. Time-course experiments indicated that both E2-2,3-Q- and E2-3,4-Q-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter. Additionally, with 2,3,7,8-tetrachlorodibenzo-p-dioxin pretreatment (10 nM for 72 h), production of estrogen quinone-derived protein adducts was also detected in human MCF-7 breast cancer cells exposed to estrogen (1 μM for 9 h). Co-treatment of Ro 41-0960, a catechol-o-methyl transferase inhibitor, further enhanced the production of estrogen quinone-derived adducts in all cases. When we investigated the levels of estrogen quinone-derived adducts in human serum Alb derived from female blood donors in Taiwan, cysteinyl adducts of E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb were detected in all subjects (n=10). The median levels of E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb in human subjects were estimated to be 398 (range 240-807) and 39.0 (range 0.3-101) (pmol/g), respectively. We noticed that levels of E2-2,3-Q-4-S-Alb were significantly correlated with those of E2-3,4-Q-2-S-Alb (correlation coefficient r = 0.684, p&amp;lt;0.05). (This work was supported by the National Science Council, Taiwan, through grants NSC95-2314-B-005-001, and NSC97-2314-B-005-001). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4712.

A Hapten Generated from an Oxidation Fragment of Docosahexaenoic Acid Is Sufficient to Initiate Age-Related Macular Degeneration

The protein adduct carboxyethylpyrrole (CEP) is present in age-related macular degeneration (AMD) eye tissue and in the blood of AMD patients at higher levels than found in age-matched non-AMD tissues. Autoantibodies to CEP are also higher in AMD blood samples than in controls. To test the hypothesis that this hapten is causally involved in initiating an inflammatory response in AMD, we immunized C57BL/6J mice with mouse serum albumin (MSA) adducted with CEP. Immunized mice develop antibodies to CEP, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, show decreased a- and b-wave amplitudes in response to light, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. Inflammatory cells are present in the region of lesions and may be actively involved in the pathology observed. We conclude that early immunization of mice with CEP-adducted MSA sensitizes these animals to the ongoing production of CEP adducts in the outer retina where DHA is abundant and the conditions for oxidative damage are permissive. In response to this early sensitization, the immune system mounts a complement-mediated attack on the cells of the outer retina where CEP adducts are formed. This animal model for AMD is the first that was developed from an inflammatory signal discovered in eye tissue and blood from AMD patients. It provides a novel opportunity for dissecting the early pathology of AMD and the immune response contributing to this disorder. The availability of a mouse with a mechanistically based AMD-like disease that progresses rapidly is highly desirable. Such a model will allow for the efficient preclinical testing of the much-needed therapeutics quickly and inexpensively.

Systems Analysis of Protein Modification and Cellular Responses Induced by Electrophile Stress

Biological electrophiles result from oxidative metabolism of exogenous compounds or endogenous cellular constituents, and they contribute to pathophysiologies such as toxicity and carcinogenicity. The chemical toxicology of electrophiles is dominated by covalent addition to intracellular nucleophiles. Reaction with DNA leads to the production of adducts that block replication or induce mutations. The chemistry and biology of electrophile−DNA reactions have been extensively studied, providing in many cases a detailed understanding of the relation between adduct structure and mutational consequences. By contrast, the linkage between protein modification and cellular response is poorly understood.In this Account, we describe our efforts to define the chemistry of protein modification and its biological consequences using lipid-derived α,β-unsaturated aldehydes as model electrophiles. In our global approach, two large data sets are analyzed: one represents the identity of proteins modified over a wide range of electrophile concentrations, and the second comprises changes in gene expression observed under similar conditions. Informatics tools show theoretical connections based primarily on transcription factors hypothetically shared between the two data sets, downstream of adducted proteins and upstream of affected genes. This method highlights potential electrophile-sensitive signaling pathways and transcriptional processes for further evaluation.Peroxidation of cellular phospholipids generates a complex mixture of both membrane-bound and diffusible electrophiles. The latter include reactive species such as malondialdehyde, 4-oxononenal, and 4-hydroxynonenal (HNE). Enriching HNE-adducted proteins for proteomic analysis was a technical challenge, solved with click chemistry that generated biotin-tagged protein adducts. For this purpose, HNE analogues bearing terminal azide or alkyne functionalities were synthesized. Cellular lysates were first exposed to a single type of HNE analogue (azido- or alkynyl-HNE), and then click reactions were performed against the cognate alkynyl- and azido-biotin derivative. The resulting biotin-labeled proteins were captured and enriched over a streptavidin matrix for subsequent mass spectrometric analysis. We thereby identified a multitude of HNE targets. Simultaneous microarray analysis of changes in gene expression triggered by HNE also produced an abundance of data. Functional analysis of both data sets generated the hypothesis that an important pathway of cellular response derives from electrophile modification of protein chaperones, resulting in the release of transcription factors that are their clients. Informatic analysis of the protein modification and microarray data sets identified several transcription factors as potential mediators of the cellular response to HNE-adducted proteins. Among these, heat shock factor 1 (HSF1) was confirmed as a sensitive and robust effector of HNE-induced changes in gene expression. Activation of HSF1 appears, in part, to be mediated by the electrophilic adduction of Hsp70 and Hsp90, which normally maintain HSF1 in an inactive cytosolic complex.The identification of HSF1 as a mediator of biological effects downstream of HSF1 has provided new opportunities for research, illustrating the potential of our systems-based approach. Accordingly, we characterized HSF1-mediated gene expression in protecting against electrophile-induced toxicity. Among the genes induced by HSF1, Bcl-2- associated athanogene 3 (BAG3) is notable for its actions in promoting cell survival through stabilization of antiapoptotic Bcl-2 proteins, appearing to have a critical role in mediating cellular protection against electrophile-induced death.

Gold(I)-Catalyzed Tandem Reactions Initiated by Hydroamination of Alkynyl Carbamates: Application to the Synthesis of Nitidine

As a convenient and direct synthesis of 1,2-dihydroisoquinolines, the gold(I)-catalyzed intramolecular hydroamination of (2-alkynyl)benzyl carbamates has been developed. The reaction with cationic gold(I) complex [AuCl(PPh(3))/AgNTf(2)] proceeded at room temperature, giving the desired 6-endo adducts. The addition of alcohol efficiently promoted the reaction, and the amount of the catalyst could be reduced to 1 mol %. However, the alkynes bearing either an electron-deficient aryl group or an alkyl group resulted in predominant production of 5-exo adducts. In such cases, use of a bulky gold catalyst, AuCl[(o-biPh)((t)Bu)(2)P]Cl/AgNTf(2), improved the regioselectivity, giving the 6-endo adducts in better yields. Furthermore, the hydroamination of alkynyl carbamates bearing an acetal or enone was successfully applied to the concise synthesis of tetracyclic heterocycles such as nitidine via the single catalyst-mediated tandem cyclization which consists of a condensation or a Michael addition of the resulting enecarbamates.

Trimethylchlorosilane-promoted aza-Mannich reaction of enecarbamates and aldimines

A trimethylchlorosilane-promoted aza-Mannich reaction is reported utilizing enecarbamates as the nucleophile and aromatic N-Boc aldimines as the electrophile. A variety of nucleophiles and electrophiles are tolerated by the reaction conditions, delivering the adduct products in excellent yields with high E-stereoselectivities.

Formation of γ-ketoaldehyde–protein adducts during ethanol-induced liver injury in mice

Ethanol metabolism promotes the formation of a variety of reactive aldehydes in the liver. These aldehydes can rapidly form covalent protein adducts. Accumulating evidence indicates that these protein adducts may contribute to ethanol-mediated liver injury. Overproduction of γ-ketoaldehydes, levuglandins (LGs) and isolevuglandins, is implicated in the pathogenesis of several chronic inflammatory diseases. γ-Ketoaldehydes can form protein adducts orders of magnitude more quickly than 4-hydroxynonenal (4-HNE) or malondialdehyde. We hypothesized that ethanol-induced oxidative stress in vivo results in overproduction of LGE 2– and iso[4]LGE 2–protein adducts in mouse liver. Female C57BL/6 mice were allowed free access to an ethanol-containing diet for up to 39 days or pair-fed control diets. Pathological markers of ethanol-induced hepatic injury including serum alanine aminotransferase, hepatic triglyceride, and CYP2E1 were elevated in response to ethanol feeding. Ethanol-induced formation of iso[4]LGE 2–, LGE 2–, and 4-HNE–protein adducts in mouse liver was dependent on both dose and duration of ethanol feeding. Deficiency of cyclooxygenase 1 or 2 did not prevent ethanol-induced iso[4]LGE 2 or LGE 2 adducts in the liver, but adduct formation was reduced in both TNFR1- and CYP2E1-deficient mice. In summary, ethanol feeding enhanced γ-ketoaldehyde–protein adduct production via a TNFR1/CYP2E1-dependent, but cyclooxygenase-independent, mechanism in mouse liver.

Investigation of the cumulative tissue doses of naphthoquinones in human serum using protein adducts as biomarker of exposure

Both 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are reactive metabolites of naphthalene that are thought to be responsible for the naphthalene-induced cytotoxicity and genotoxicity. The aim of this study was to investigate the cumulative tissue dose of 1,2-NPQ and 1,4-NPQ in human serum derived from blood donors in Taiwan via measurements of albumin adducts by a methodology, which employs trifluoroacetic acid anhydride and methanesulfonic acid to selectively cleave cysteinyl adducts on proteins. Both 1,2-NPQ and 1,4-NPQ adducts were detected in all male and female subjects ( n = 22). The median levels of 1,2-NPQ adduct in human subjects were estimated to be 268 (range 139–857) and 203 (range 128–1352) (pmol/g) in male ( n = 11) and female ( n = 11) subjects, respectively. In contrast, the median levels of 1,4-NPQ adduct were estimated to be 45.0 (range 22.0–117) and 38.9 (range 21.5–172) (pmol/g) in male and female subjects, respectively. We noticed that levels of 1,2-NPQ adduct were significantly correlated with those of 1,4-NPQ adduct (correlation coefficient r = 0.643, p < 0.01). Results from in vitro experiments confirmed that the production of naphthoquinones-derived adducts on serum albumin increased with increased concentration of naphthoquinones (0–100 μM). Linear relationships were observed over the range of concentration. Time-course experiments suggested that both 1,2-NPQ and 1,4-NPQ-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter. The reaction rate constant analyses indicated that the second-order rate constants, representing in vitro reactions between naphthoquinones and cysteine residues of serum albumin, were estimated to be 0.0044/0.0002 L(g protein) −1 h −1, respectively. Overall, the cumulative tissue doses of 1,4-NPQ (217–316 nM h) in male and female subjects were ∼3-fold greater than those of 1,2-NPQ (76–98 nM h) in the study population. The initial concentrations of serum 1,2-NPQ and 1,4-NPQ in the study population were estimated to be between 145–188 and 807–1175 nM, respectively. We conclude that the relatively large amounts of naphthoquinones present in human serum may point to toxicological consequences.

Inhibition of mutagenic PhIP formation by epigallocatechin gallate via scavenging of phenylacetaldehyde

Chemical model investigation showed that both epigallocatechin gallate (EGCG) and its peracetate, which has all the hydroxyl groups acetylated, effectively reduced the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine found in foods. Mechanistic study was subsequently carried out to characterize the probable inhibitory mechanism involved. GC-MS analysis showed that EGCG in only one-fourth molar quantity of phenylalanine reduced formation of phenylacetaldehyde, a key PhIP intermediate by nearly 90%. Its peracetate also showed similar inhibitory activity. This further supported the existence of an antioxidant-independent mechanism contributing to the inhibition of PhIP formation by EGCG. Subsequent LC-MS analyses of samples from a wide range of model systems consisting of PhIP precursors showed the generation of characteristic analytes with molecular weight corresponding to the sum of EGCG and phenylalanine fragment(s) only in models where phenylalanine and EGCG were simultaneously present. An isotope-labeling study revealed that these analytes all contained fragment(s) of phenylalanine origin. Direct reaction employing phenylacetaldehyde and EGCG further confirmed the capability of EGCG to form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. Finally, an investigation of the time course of the generation of postulated adduction products supported EGCG as an effective inhibitor of PhIP formation in prolonged heating processes.