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Many degenerative or pathological processes, such as aging, cancer and coronary heart disease, are related to reactive oxygen species (ROS) and radical-mediated reactions. We examined the effectiveness of aged garlic extract (AGE), a garlic preparation rich in water-soluble cysteinyl moieties, for protection of cells from ROS produced by 6-hydroxy-dopamine (6-OHDA) using human neuroblastoma SH-SY5Y cells. Concomitant treatment of cells with AGE (2 and 4 mg/ml) showed the dose-dependent protective effect on the cell death induced by 6-OHDA. In addition, the AGE treatment significantly suppressed the increase of ROS generation by 6-OHDA. Furthermore, the protective effect of AGE was accompanied by activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the increase of mRNAs of heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. These two enzymes are important in the cellular antioxidant system. These results indicated that AGE protected cells from ROS damage by not only capturing ROS directly but also activating the cellular antioxidant system by stimulating antioxidant gene expression via the Nrf2-ARE pathway. The present study suggested that AGE may be useful for prevention and treatment of cell damage caused by ROS.

Background: We have been searching effective compounds that can stimulate the growth and differentiation of nerve cells. We found previously that fulleren derivatives enhanced induction of morphological differentiation with neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. In the course of our further search for other effective compounds, we found the aged garlic extract (AGE) has the activity similar to fulleren. Methods: PC12 cells were used to examine the effectiveness of test compound. Results: AGE enhanced the stimulating effect of NGF to induce morphological differentiation with neurite outgrowth in PC12 cells. In order to examine the active constituents of AGE, it was fractionated into several components. The activity was mainly localized in the F1 fraction that contains low molecular weight polar compounds. S-Allylmercaptocysteine (SAMC) is one of the sulfur components of AGE present in F1 fraction and found to exhibit the enhancing effect similar to AGE. Conclusion: AGE had the ability to induce morphological differentiation with neurite outgrowth in NGF-treated PC12 cell and SAMC was one of its active constituents.

Water-soluble [60]fullerene (C 60 ) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C 60 derivatives together with some reported ones were tested. Among the compounds tested, C 60 /(γ-CyD) 2 , C 60 -bis(γ-CyD) ( 5 ) containing C 60 -mono(γ-CyD) ( 5′ ), and C 60 /PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.

We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on β-elimination of the phosphate group from serine/threonine phosphorylation sites under alkaline conditions, followed by Michael addition reaction with N-(2-mercaptoethyl)-6-methylnicotinamide (MEMN). As a result of the derivatization reaction, the negatively charged phosphate group is substituted with the nicotinoyl moiety to improve the ionization efficiency of the derivatized peptide. The combination of d3-labeled MEMN (d3-MEMN) and MEMN (d0-MEMN) generates a 3 Da mass difference between d3-MEMN-labeled and d0-MEMN-labeled peptides, which is a useful signature for the identification of peptides containing serine/threonine phosphorylation sites in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum. Moreover, the mass difference is useful for the quantitative analysis of serine/threonine phosphorylation in proteins. In this paper, we describe the synthesis of d0/d3-labeled MEMN and an application of our approach to model peptides and proteins. Copyright © 2008 John Wiley & Sons, Ltd.

Regulatory policies designed to reduce the health risk of environmental and/or synthetic chemicals generally aim for zero or negligible levels. Foods, on the other hand, especially those with a long history in the human diet, have been treated as essentially safe, even though they too contain various chemicals including nutrients. The recent debate on the presence in food of acrylamide, a possible human carcinogen, is likely to shake up the traditional paradigm held by regulatory agencies on chemical health risks. The current stance on the safety of acrylamide in food seems to be an extension of the traditional approach to assessment of environmental and/or synthetic chemicals. However, even foods which have long been a part of the human diet contain components that do not necessarily meet the safety margins applied to environmental and/or synthetic chemicals. In the future, a greater understanding of the effect of these agents on biological systems as well as the development of analytical methods for testing will result in many questions being raised concerning chemicals in foods, such as acrylamide which is under scrutiny today. Regulatory policies currently employ various standards for controlling chemical risk. These standards are dependent upon the labeling of the chemical in question, e.g., whether carcinogenic or non-carcinogenic, synthetic or natural, or whether a food or industrial chemical. Regardless of labeling, all chemicals to which we are exposed should be evaluated on an equal footing. Then, according to the level of the identified health risk, regulations could or could not be applied based on local circumstances, e.g., public acceptance, voluntary risk vs. involuntary risk, etc. In order to create a standardized system for chemical risk assessment, the introduction of uniform measures is essential. Loss of life expectancy (LLE) is one possible measure to assess chemical health risk. When LLE has been used, animal toxicity data have indicated that an ad libitum diet intake has considerably more impact on health risk than the acrylamide concentration of the ingested food. Reassessing the health effects of chemicals with a system of uniform measures could reveal many risks that need to be preferentially addressed above and beyond keeping minor toxicants to zero or negligible levels. Recognition of such risks may result in changes that conflict with existing regulations. In any case, whether consciously or unconsciously, people have always been exposed to a certain degree of chemical risk in their daily life. Based on the premise that the public can accept some degree of chemical risk in balance with other risks in their lives, regulatory bodies should be able to take a flexible and effective approach. In order to efficiently and comprehensively maximize the protection of our health against potential harm from chemicals using limited public resources, it is now time for regulatory agencies to restructure their policy frameworks across categories for controlling chemical health risks.

To develop novel PPARγ ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which are designed based on the structures of rosiglitazone and 15d-PGJ2. Among these compounds, compound 9 was found to be as potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARγ protein where the alkyl chain of 15d-PGJ2 is expected to be located.

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i). substrate (acetyl lysine) analogues (compounds 3-7), (ii). analogues bearing various functional groups expected to chelate zinc ion (compounds 8-15), and (iii). analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16-18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.

Reaction of 7-amino-9-ethylguaninium chloride with lead(IV) acetate (LTA) in MeOH yielded 8-aza-9-ethylguanine. Similarly, the reaction of 1-amino-3-methylbenzimidazolium chloride or its substituted derivatives (6-methyl, 5,6-dimethyl and 5-nitro) with LTA gave the corresponding 1-methyl-1H-benzotriazole (or 1-methyl-2-azabenzimidazole) derivatives along with N-methylformanilide derivatives.

Reactions of 7-aminoadenines with 2,4-pentanedione yielded pyridazino[6,1-f]purines and 5-pyrazolylpyrimidines. The structure of the latter was identified by X-ray analysis. Reaction of 9-amino-N6-methyladenine with 2,4-pentanedione gave pyridazino[1,6-e]purine and Schiff base products. Possible reaction mechanisms are discussed.

Aryl radicals and arenediazonium ions are suspected to react with cellular DNA, resulting in C8-arylguanine adducts. 8-Phenyl-2'-deoxyguanosine (8-PhdG) was synthesized as a model adduct by reacting dG with benzenediazonium chloride and incorporated into oligodeoxynucleotides using phosphoramidite techniques. A site-specifically modified oligodeoxynucleotide containing a single 8-PhdG was then used as a template for primer extension reactions catalyzed by the intact (exo+) or 3'-->5' exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha (pol alpha). Although primer extensions catalyzed by the Klenow fragments were retarded at the position of 8-PhdG, most of the primer extension passed the lesion to form the fully extended products. In contrast, primer extensions catalyzed by pol alpha were strongly blocked opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-PhdG. The exo- Klenow fragment incorporated primarily dCMP, the correct base, opposite 8-PhdG, along with small amounts of incorporation of dAMP. Two-base deletions were also observed. In contrast, the exo+ Klenow fragment incorporated dCMP opposite the lesion. When pol alpha was used, 8-PhdG promoted small amounts of misincorporation of dAMP and dGMP as well as one- and two-base deletions. The duplex containing 8-PhdG.dG was thermally and thermodynamically more stable than dG.dG. The duplex containing 8-PhdG.dA was thermodynamically more stable than dG.dA. We conclude that 8-PhdG is a weak miscoding lesion, capable of generating G-->T and G-->C transversions and deletions in cells.

To investigate the substrate specificity of human O6-methylguanine-DNA methyltransferase (MGMT) for O6-benzylguanine (6BG) derivatives incorporated in oligodeoxynucleotides, we prepared 25-mer lengths of sequences containing various 6BG derivatives and their related compounds and then measured the ability of these derivatives to inactivate MGMT in vitro. Oligodeoxynucleotides containing a 6BG, O6-(2-fluorobenzyl)guanine (2F-6BG), O6-(3-fluorobenzyl)guanine (3F-6BG), O6-(4-fluorobenzyl)guanine (4F-6BG), O6-benzylhypoxanthine (6BH), or O6-methylguanine (6MG) were all good substrates for MGMT, and no obvious differences were observed among them. Oligodeoxynucleotides containing N2-isobutyrylated 6BG and 6MG showed only a slightly reduced capacity for inactivating MGMT compared to N2-nonmodified forms of these derivatives. No obvious differences were observed in the corresponding double-stranded and single-stranded oligodeoxynucleotides. MGMT substrate specificity for the 6BG derivatives in the oligodeoxynucleotide was found to be quite different from that seen in our previous study [Mineura, K., et al. (1994) Int. J. Cancer 58, 706-712; (1995) Int. J. Cancer 63, 148-151. Kohda, K., et al. (1995) Biol. Pharm. Bull. 18, 424-430] and others [Moschel, R. C., et al. (1992) J. Med. Chem. 35, 4486-4491. Chae, M.Y., et al. (1994) J. Med. Chem. 37, 342-347] using the corresponding free bases. In brief, (i) 6BG, 3F-6BG, and 4F-6BG greatly inhibited human MGMT, whereas 2F-6BG, 6BH, and 6MG displayed much weaker activity; (ii) any modifications at the 2-amino group of the 6BG resulted in severe reductions in the ability to inactivate MGMT. These results obtained by the experiments using oligodeoxynucleotides and free bases suggest that human MGMT has low substrate specificity for 6BGs in oligodeoxynucleotides. Conformational changes in human MGMT which favor binding to oligodeoxynucleotides containing 6BG derivatives and the subsequent transfer of their benzyl groups may account for the difference in substrate specificity between the incorporated 6BG derivatives and their free base form.

A new preparation method was devised using beta-cyclodextrin as a mobile-phase additive in a reversed-phase high-performance liquid chromatography system. Preparative separation of the biologically active (R)-enantiomer was achieved from racemic N-methylsalsolinol. Beta-cyclodextrin was removed completely in good yield by acid extraction and solid-phase extraction. By a slight modification, this method will be applicable to the isolation of various types of biologically important enantiomers.

Several triterpenes were tested for cytotoxicity by our contrived primary screening method using resting or dormant leukemia L1210 cells after 3 d-preculture without medium change. Some triterpenes were found to be more cytotoxic toward the 3 d-precultured resting cells than toward the growing cells in a fresh medium. These triterpenes are distinguished by highly selective cytotoxicity toward the starved resting cells unlike common anticancer agents. The highest selectivity was shown by betulinic acid, the ratio of its IC50 values toward the growing versus resting cells amounting to 175. It is suggested that this selective cytotoxicity is attributable to low pH (or = 6.8) of the medium. It is noteworthy that betulinic acid is not cytotoxic at all in media of ordinary pH (or = 7.0) even after a 48-h exposure. Betulinic acid might be promising as an antitumor agent toward solid tumors because the interior pH of tumor tissues is generally lower than in normal tissues.

In the human brain, only (R)enantiomer of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline ((R)salsolinol) and N-methyl-salsolinol, a dopaminergic neurotoxin, were detected, suggesting their enzymatic biosynthesis. This paper reports the isolation and characterization of a novel enzyme, which enantio-selectively synthesizes (R)salsolinol from dopamine and acetaldehyde. Dopamine, acetaldehyde, formaldehyde and pyruvic acid were the substrates of this synthase, whereas N-methyldopamine, adrenaline, noradrenaline and L-DOPA were not. The possible function of this enzyme under physiological and pathological conditions in the brain is discussed.

Reaction of 9-substituted (methyl or benzyl) 1-aminoadenines 1 with hydrazine afforded 9-substituted 6-hydrazinopurines 2 and 1-substituted 5-ammo-4-(4-amino-1,2,4-triazol-3-yl)imidazole (4). The product ratio of 2 to 4 rose with increasing amounts of methanol used as the solvent. When the same reaction was carried out using 1,9-dimethyladenine instead of 1, compounds 2 and 4 were also obtained with N6,9-dimethyladenine. A possible mechanism for formation of 2 and 4 is discussed.

The alkaline comet assay, employing a single cell gel electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual mammalian cells. In this report, we describe a modified version of this assay which we used to assess DNA damage as a result of treating lysed cells with genotoxic and antimetabolic agents. By means of this modified assay, DNA is no longer held under the regulation of any metabolic pathway or membrane barrier. Using 3 direct-acting agents, hydrogen peroxide, N-methyl-N-nitrosourea, and bleomycin, we were able to induce increased DNA migration by both the standard and modified comet assays. In contrast, with 4-nitroquinoline 1-oxide, 5-fluorouracil, and methotrexate, which require cellular enzymatic activity to induce DNA damage, we succeeded in inducing increased DNA migration using the standard comet assay conditions only. In some cases, the modified comet assay might be helpful in analyzing chemical and biological characteristics of genotoxic agents when performed in combination with the standard comet assay.

O 6 -Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O 6 -methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O 6 -alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O 6 -fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O 6 -benzylguanine and O 6 -phenylguanine. The O 6 -(4-and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O 6 -benzylguanine. In contrast, O 6 -(2-fluorobenzyl)guanine and O 6 -phenylguanine had less of an effect on the activity. Two-hour pretreatment of O 6 -(4-and 3-fluorobenzyl)guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O 6 -(2-fluorobenzyl)guanine and O 6 -phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O 6 -fluorobenzylguanine derivatives. O 6 -Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O 6 -benzylguanine and were powerful MGMT inactivators. The chemical features of the O 6 -benzyl group are a biologically important determinant in the reaction evolution with MGMT.

O6-Benzylguanine (BG) is a potent depleter of a repair enzyme O6-alkylguanine-DNA alkyltransferase. Pretreatment of cells with BG potentiates the cytotoxicity of chloroethylating anti-cancer agents. In this study we used HeLa S3 cells to examine the cytotoxic potentiation of 39 compounds after BG pretreatment. Compounds tested included anti-cancer agents and carcinogens, and among them only the cytotoxicity of methylating, chloroethylating and fluoroethylating agents was potentiated. This is the first description of the cytotoxic potentiation of fluoroethylating agents. Potentiation ratios were found to vary even among compounds possessing the same alkylating group. By pretreatment with 10 microM of BG, the cytotoxicity of methylating agents such as N-methyl-N-nitrosourea and streptozotocine was potentiated 3.7 and 9.4 fold, respectively. For chloroethylating agents, the potentiation ratios were 3.5 for N-chloroethyl-N-nitrosourea, 8.6 for N-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N'-(2-chloroethyl)-N'-nitroso urea (ACNU), 2.2 for N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), 3.0 for N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU) and 5.2 for chloroethyl methanesulfonate. With respect to fluoroethylating agents, the potentiation ratios were 7.2 for N-fluoroethyl-N-nitrosourea, 2.0 for N-cyclohexyl-N'-fluoroethyl-N'-nitrosourea and 5.5 for fluoroethyl methanesulfonate. No effect was observed with the bromoethylating agent, N-bromoethyl-N-nitrosourea. There was no potentiation of the cytotoxicity of anti-cancer agents such as mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5FU), bleomycin (BLM), prednisolone, camptothecin, etoposide, methotrexate or vinblastine. A possible mechanism for the cytotoxic potentiation of the test compounds by BG pretreatment is discussed.

Amination of adenine with H2N-O-SO3H in alkaline media afforded 1-, 3-, 7-and 9-aminoadenine isomers at a ratio of about 1:1:3:1. In neutral media, the product ratio of the isomers changed to about 3:1:1:0. These results were different from the regioselectivity obtained by methylation of adenine with dimethyl sulfate under similar conditions. Amination of adenine with dinitrophenoxyamine in DMF gave 1-aminoadenine as the main product and this regioselectivity was also different from that of methylation with cH3I. Chemical characteristics of these N-amino adenines are described. This paper is dedicated to Professor Emeritus Yoshihisa Mizuno of Hokkaido University on the occasion of his 75th birthday.

DNA strand breakage induced in cultured cells by bleomycin (BLM) was remarkably enhanced in the presence of glycerol or polyvinyl alcohol which produces an enhancement effect on BLM cytotoxicity, but not in the presence of alcohols such as methanol, ethanol, or polyethylene glycol which do not produce enhanced cytotoxicity. The comet assay, a useful analytical method for evaluating DNA strand breakage, clearly demonstrated that combinations of BLM and polyhydric alcohols induced serious DNA damage in the whole cell populations compared with those induced by treatment with BLM alone. The comet assay also successfully showed distributions of cell populations with various degrees of DNA damage. These results suggested that an increase in DNA damage induced in the presence of polyhydric alcohols might be responsible for the enhancement of BLM cytotoxicity.

The mechanism of formation of N2-benzylguanine in the reaction of 2-amino-6-chloropurine with a large excess (12-13 molar eq) of sodium benzyl oxide in benzyl alcohol at 130°C was studied, N2, O6-Dibenzylguanine, a reaction intermediate, was isolated and a possible mechanism for its formation is discussed. Furthermore, using this sodium benzyl oxide system, benzylation at the amino group of nucleic acid bases was facilitated.

Activity of O 6 -methylguanine-DNA methyltransferase (MGMT) is well related with drug resistance of tumor cells to chloroethylnitrosoureas (CENUs), because MGMT removes CENU-induced O 6 -alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. Inactivation of MGMT is a feasible way to overcome MGMT-related resistance of tumor cells to CENUs. O 6 -Benzylguanine is known to be a strong depleter of MGMT. We previously reported the potentiation effect of O 6 -arylmethylguanine derivatives on cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), a CENU, for HeLa S3 cells. In this study, we tested the activity of these O 6 -arylmethylguanine derivatives as depleters of MGMT using HeLa S3 cell-free extract. The O 6 -arylmethylguanine derivatives tested were O 6 -benzylguanine (1), O 6 -(4-, 3-, and 2-fluorobenzyl)guanines (2-4), O 6 -(4, 3-, and 2-trifluoromethylbenzyl)guanines (5-7), O 6 -(4-, 3-, and 2-pyridylmethyl)guanines (8-10), and O 6 -(2- and 1-naphtylmethyl)guanines (11,12). Among these, compounds 1-3, 5, 8, 9 and II showed a strong MGMT depletion activity, whereas compounds 4, 6, 7, 10 and 12 were inactive. These inactive compounds, except for 6, have a substituent at the α position of the benzyl group (4, 7, 12) or are an α nitrogen analogue of 1 (10). There was a good relation (r = -0.856, p < 0.001) between the MGMT depletion activity of O 6 -arylmethylguanine derivatives and their potentiation activity of ACNU cytotoxicity. These results suggest that the α position of the benzyl group plays an important role in the interaction of O 6 -arylmethylguanine derivatives with MGMT to result in the inactivation of MGMT. Potent MGMT inactivators (1-3, 5, 8, 9, 11) sensitize tumor cells to CENU chemotherapy.

Tilivalline (TV) possesses a pyrrolo[1,4]benzodiazepine nucleus and is cytotoxic toward mammalian cells. The 11-β-cyano TV analogue (1) is about one hundred times more cytotoxic to mouse leukemia L1210 cells than TV itself. In contrast, the 11-α-cyano TV analogue (2), an epimer of 1, has only about one-hundredth the cytotoxicity of 1. It was found that epimerization proceeded between 1 and 2 under physiological conditions, and the cytotoxicity of 2 is thought to be caused mainly by 1 that was formed from 2 during incubation in medium.

It was reported recently that monomeric O6-benzylguanine (1) acts as an alternative substrate for a DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), and that therefore pretreatment of cells with 1 induces depletion of AGT resulting in an enhanced cytotoxic response to alkylating antitumor agents. In order to study the interaction of O6-benzylguanine derivatives with AGT and to obtain greater AGT depletion, we synthesized the following O6-arylmethylguanine derivatives and related compounds: O6-(4-, 3- and 2-fluorobenzyl)guanines (2, 3, 4), O6-(4-, 3- and 2-trifluoromethylbenzyl)guanines (5, 6, 7), O6-(4-, 3- and 2-pyridylmethyl)guanines (8, 9, 10), O6-(2- and 1-naphthylmethyl)guanines (11, 12), O6-biphenylmethylguanine (13), S and Se analogues of O6-benzylguanine (14, 15) and O6-phenylguanine (16). Ten of these are new compounds. All these compounds were tested for their potentiation of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosou rea (ACNU) cytotoxicity using HeLa S3 and C6-1 cells. Compounds 2, 3, 5, 8, 9, 11 and 13 were active, as was 1. Compounds 7 and 12, with a substituent at the alpha position of the benzyl group, and compound 10, the alpha-nitrogen analogue of 1, were almost completely devoid of potentiating activity. These results suggest that the alpha-position of the O6-benzyl group plays an important role in the interaction of O6-benzylguanines with AGT. Of the other compounds, 4 and 6 exhibited very weak activity and 14, 15 and 16 were inactive. Possible reasons for these differences in activity are discussed in relation to the biomimetic dealkylation rates of O6-benzylguanine derivatives and the chemical characteristics of their substituents.

cDNA for mouse O 6 -methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state. Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells. A single cell of NIH3T3 contained 1.8x10 4 molecules of the methyltransferase protein. When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus. Using double-stranded oligomers containing a single O 6 -methylguanine or O 4 -methylthymine at predetermined sites, the mouse enzyme repaired 06-methylguanine and O 4 -methylthymine, at an almost equal efficiency. In the LacZ reversion assay, MNNG-induced A :T to G :C as well as G :C to A :T transition mutations were efficiently suppressed by the function of mouse methyltransferase, in vivo.

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N(1)-acetylspermidine (N(1)AcSpd), N(8)-acetylspermidine (N(8)AcSpd), N(1)-acetylspermine, N(1),N(8)-diacetylspermidine, and N(1),N(12)-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N(1)AcSpd and N(8)AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with (13)C(2)-N(1)AcSpd and (13)C(2)-N(8)AcSpd which have the (13)C(2)-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N(1)-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N(1)-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-(15)N(3)]-N(1)-acetylspermine and [1,4,8-(15)N(3)]spermidine ((15)N(3)-Spd), respectively; for SMO, [1,4,8,12-(15)N(4)]spermine and (15)N(3)-Spd, respectively; and for SSAT, (15)N(3)-Spd and [1,4,8-(15)N(3)]-N(1)-acetylspermidine, respectively.

O6-Alkylguanine derivatives are well known as chemical modulators of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Depletion of the enzyme by these derivatives leads to increase sensitivity of tumor cells to chloroethylnitrosoureas. We tested the effect of O6-methylguanine, O6-benzylguanine, O6-(p-methylbenzyl)guanine, O6-(p-chlorobenzyl)guanine, O6-(p-methoxybenzyl)guanine, O6-methylhypoxanthine and O6-benzylhypoxanthine on the sensitivity of tumor cell lines to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) using a colorimetric cytotoxicity assay. The sensitivity of MGMT-proficient tumor cells including HeLA S3, C6-1, C6-2/ACNU, U-138 MG and U-373 MG cells was greatly enhanced by 2 hr pretreatment of 10-100 microM O6-benzylguanine, O6-(p-methylbenzyl)guanine and O6-(p-chlorobenzyl)guanine, but not by O6-methylguanine or O6-methylhypoxanthine. O6-(p-methylbenzyl)guanine moderately sensitized the 2 cell lines, HeLa S3 and C6-1, tested in our study to ACNU cytotoxicity. O6-Benzylhypoxanthine at the high concentration (100 microM) sensitized, to some extent, 3 MGMT-proficient cell lines. Lesser degrees of enhancement by the O6-benzylguanine derivatives were noted in MGMT-deficient tumor cells. Biological effects of O6-alkylguanine derivatives on enhancing ACNU cytotoxicity of tumor cells suggest that the exocyclic 2-amino and O6-benzyl groups in O6-benzylguanine skeleton are both essential for the inhibition of MGMT activity.

Reactions of 1-aminoadenines (2) with NH2OH gave adenine 1-oxides (6). Alkaline treatment of 2 afforded 5-amino-4-(1, 2, 4-triazol-3-yl)imidazoles (3), which were converted to 3H-imidazo[4, 5-e][1, 2, 4]triazolo[1, 5-c][1, 2, 3]triazines (5), aza analogues of 3H-[1, 2, 4]triazolo[3, 2-i]purines (4), by treatment with NaNO2.

In order to investigate the chemical characteristics of O6-methylguanines, the substrate of O6-methylguanine-DNA methyltransferase, cis[PtCl(NH3)2(O6,9-dimethylguanine-7)]NO3, a 1:1 complex of cis-platin with O6,9-dimethylguanine, was synthesized and its demethylation rate, pKa, X-ray structure and cytotoxicity were determined.

The effect of fatty acids on 8-hydroxydeoxyguanosine (8-OH-dG) formation in calf thymus DNA treated with bleomycin (BLM)-Fe(II) in vitro was studied. The formation of 8-OH-dG was greatly reduced in the presence of stearic acid (18:0) or oleic acid (18:1), however, it increased to the control level with an increase in the number of unsaturated bonds of fatty acids (18:2, 18:3). This increase in 8-OH-dG formation may have resulted from the indirect oxidation of DNA by the peroxidized unsaturated fatty acids formed by BLM-Fe(II). The inhibitory effect of saturated fatty acids on 8-OH-dG formation was observed with those possessing more than 12 methylene chains. Stearic acid also inhibited peroxidation of unsaturated fatty acids (18:2, 18:3) treated with BLM-Fe(II), however, it had no effect on ribose degradation of DNA treated with BLM-Fe(II). These results suggest that different active oxygen species are probably involved in DNA degradation and in formation of 8-OH-dG in DNA and of peroxidized products in fatty acids.

Syntheses of the positional isomers of N -aminopyrimidine bases (1- and 3-amino derivatives of uracil, thymine, and cytosine), 3-aminopyrimidine deoxyribonucleosides (3-amino derivatives of deoxyuridine, thymidine, deoxycytidine), and related compounds are reported. Physicochemical properties of these N -aminopyrimidines are described.

Electrophilic amination of four 9-ethylguanine derivatives and seven 1-methylbenzimidazole derivatives with 2,4-dinitrophenoxyamine was carried out. N-amination proceeded at the imidazole moiety of these compounds with pKa's of more than 3.2. Treatment of these N-aminated derivatives with 0.1 N NaOH gave different results depending on their structures. Reactivity of the imidazole moiety to electrophilic amination and the reaction pathways of N-aminated compounds with alkaline treatment are discussed.

Syntheses of the positional isomers of N -aminopyrimidine bases (1- and 3-amino derivatives of uracil, thymine, and cytosine), 3-aminopyrimidine deoxyribonucleosides (3-amino derivatives of deoxyuridine, thymidine, deoxycytidine), and related compounds are reported. Physicochemical properties of these N -aminopyrimidines are described.

Electrophilic amination of four 9-ethylguanine derivatives and seven 1-methylbenzimidazole derivatives with 2,4-dinitrophenoxyamine was carried out. N-amination proceeded at the imidazole moiety of these compounds with pKa's of more than 3.2. Treatment of these N-aminated derivatives with 0.1 N NaOH gave different results depending on their structures. Reactivity of the imidazole moiety to electrophilic amination and the reaction pathways of N-aminated compounds with alkaline treatment are discussed.

Reactions of 1-aminoadenines (2) with NH2OH gave adenine 1-oxides (6). Alkaline treatment of 2 afforded 5-amino-4-(1, 2, 4-triazol-3-yl)imidazoles (3), which were converted to 3H-imidazo[4, 5-e][1, 2, 4]triazolo[1, 5-c][1, 2, 3]triazines (5), aza analogues of 3H-[1, 2, 4]triazolo[3, 2-i]purines (4), by treatment with NaNO2.

Reaction of 9-substituted (methyl or benzyl) 1-aminoadenines 1 with hydrazine afforded 9-substituted 6-hydrazinopurines 2 and 1-substituted 5-ammo-4-(4-amino-1,2,4-triazol-3-yl)imidazole (4). The product ratio of 2 to 4 rose with increasing amounts of methanol used as the solvent. When the same reaction was carried out using 1,9-dimethyladenine instead of 1, compounds 2 and 4 were also obtained with N6,9-dimethyladenine. A possible mechanism for formation of 2 and 4 is discussed.

Oxidation of seven 1-methylbenzimidazole (MBI) derivatives (with pKa's ranging from 1.6 to 6.0) was carried out with m-chloroperoxybenzoic acid and structures of the products formed were identified. (Condensed benzene moiety-hydroxylated)-2-(m-chlorobenzyloxy)-MBIs and 2-oxo-MBIs were obtained from MBIs with pKa's of more than 5.6 and about 3.3, respectively.

Reaction of 7-amino-9-ethylguaninium chloride with lead(IV) acetate (LTA) in MeOH yielded 8-aza-9-ethylguanine. Similarly, the reaction of 1-amino-3-methylbenzimidazolium chloride or its substituted derivatives (6-methyl, 5,6-dimethyl and 5-nitro) with LTA gave the corresponding 1-methyl-1H-benzotriazole (or 1-methyl-2-azabenzimidazole) derivatives along with N-methylformanilide derivatives.

Protein nitration at tyrosine residues proceeds to form 3-nitrotyrosine; this product is today used as a biomarker of nitrative stress. We have developed an efficient method with which to identify nitrated peptides and to quantify protein nitration levels in different biological samples by a combination of the chemical derivatization of nitrotyrosine-containing peptides and mass spectrometry. Our strategy includes: 1) the protection of both N-terminal amines and epsilon-amines of lysine residues by acetylation with (13)C(0)/(13)C(4)- or D(0)/D(6)-acetic anhydride; 2) the reduction of nitrotyrosine to aminotyrosine with sodium hydrosulfite; and 3) the derivatization of aminotyrosine with 1-(6-methyl[D(0)/D(3)]nicotinoyloxy)succinimide. The utility of our method is demonstrated with nitrotyrosine-containing angiotensin II and bovine serum albumin as the model compounds.

A method for the quantification of acetylpolyamines, N(1),N(12)-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N(1),N(8)-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using (15)N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of alpha,omega-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N(1)-acetylspermidine and N(8)-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).

Human pancreatic tumor cell lines – AsPC-1, PANC-1, MIA paca2, KP-1 and KP-59 cells – can be induced to differentiate into pancreatic hormone-producing cells by brief trypsin treatment and subsequent culture in a serum-free, chemically defined medium. During culture, AsPC-1 cells formed cell clusters resembling the pancreatic islets, expressed genes associated with the pancreatic development and produced glucagon but not insulin. When PANC-1, MIA paca2, KP-1 and KP-59 cells were treated and cultured the same way, they underwent similar morphological changes and produced insulin and glucagon. We used these systems to identify intracellular regulatory molecules involved in the conversion of pancreatic tumor cells into glucagon-producing cells. We found that the expression of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase, was increased and its localization was altered from the nucleus to the cytoplasm during AsPC-1 cell differentiation. Transient transfection of AsPC-1 cells with AZ1 siRNA resulted in inhibition of the morphological and functional cell differentiation as well as the specific suppression of AZ1 expression. By contrast, constitutive overexpression of AZ1 in AsPC-1 cells led to the enhancement of glucagon production. We also found that PANC-1 cells reduced the expression of glucagon mRNA when treated with AZ1 siRNA. These results suggested that AZ1 was necessary for the conversion of pancreatic tumor cells into glucagon-producing cells. Glucagon production in AsPC-1 cells was not affected by addition of putrescine, suggesting that the polyamines were not directly involved in the AZ1-mediated conversion of pancreatic tumor cells to differentiated state.

Based on our recent finding regarding diverse biological activities of natural lignified materials, synthetic dehydrogenation homo- and copolymers were prepared using 3 p-hydroxylated cinnamic acids and coniferyl alcohol in order to explore the role of lignin skeleton in the activities displayed by natural lignins. The synthetic polymers stimulated polymorphonuclear cell iodination as potently as the natural lignifined materials.

Bacterial O 6 -alkylguanine-DNA alkyltransferase (AGT) removes alkyl group from O 6 -alkylguanine and O 4 -alkylthymine residues in DNA, both of which are considered to be DNA damages most related to the induction of cancer and/or mutation. The repair process involves alkyl-transfer of an O -alkyl group to the active site of the enzyme, where an SH-group of cysteine residue plays the role of alkyl acceptor. In order to elucidate the chemical characteristics of substrates for this enzyme, dealkylation rates of O 6 -alkyldeoxyguanosine, O 4 -alkylthymidine and related compounds were measured using an alkyltransfer system. Thiophenol-triethylamine system was employed as an alkyl acceptor and twenty-one O -alkyl compounds were tested. Dealkylation proceeded with pseudo first order kinetics. The half-life of O 6 -methyldeoxyguanosine (MedG) was 122 h and no remarkable dependence on N-9 substituents (H, CH 3 and deoxyribose) was observed. A compound lacking 2-NH 2 group underwent demethylation about three times faster than O 6 -methylguanines did, while, a compound lacking imidazole moiety underwent demethylation about 2.5 times more slowly. The half-life of O 4 -methylthymidine (MedT) was 38 h and no remarkable dependence on N-1 (H, CH 3 and deoxyribose) and C-5 (H and CH 3 ) substituents was observed. Deethylation proceeded much more slowly than demethylation. Substitution of selenophenol for thiophenol resulted in a 4.5 times faster MedG demethylation rate. Demethylation rates were moderately correlated with values for NMR chemical shift of CH 3 group, an indicator of electron density, although the correlation curves of a series of MedG and MedT derivatives were quite different. This result suggests that some different ratedetermining factors other than electron density are playing a role. These findings may be of help in resolving the details of the mechanisms of enzymic repair by bacterial and mammalian AGT.

Treatment of calf thymus deoxyribonucleic acid (DNA) with the horseradish peroxidase-H2O2 system resulted in efficient formation of 8-hydroxydeoxyguanosine (8-OH-dG) residues. It was concluded that singlet oxygen was the reactive species involved, based on experiments using active oxygen scavengers and D2O. For 8-OH-dG formation, a higher-ordered polynucleotide structure seems to be required: double stranded DNA was a better substrate for the reactive species than single stranded DNA, and monomeric deoxyguanosine underwent C8-hydroxylation to a lesser extent.

A study of the mechanism of 8-aminoguanosine formation in the reaction of guanosine with hydroxylamine- O -sulfonic acid (HAOS) revealed that the reaction proceeds in three steps; 1. electrophilic N-7 amination by HAOS, 2. nucleophilic C-8 hydroxyamination by NH2OH accompanied by elimination of the N-7 amino group and by aromatization, and finally 3. the reduction of the C-8 hydroxyamino group by NH2OH to give 8-aminoguanosine. The significance of formation of the 7-aminoguanosine intermediate is discussed briefly in relation to the reaction of carcinogenic arylamines and arylhydroxylamines with cellular DNA.

We describe a convenient and useful method for the identification and relative quantification of proteins using light and heavy reagents, 1-(6-methylnicotinoyloxy)succinimides (6-CH3-Nic-NHS and 6-CD3-Nic-NHS, respectively). This method is based on the chemical derivatization of amino groups of tryptic peptides with these reagents, i.e., the basic moiety of the reagents thus incorporated into both the N-terminal amino group and the e-amino group of the lysine residue would improve the ionization efficiency of tryptic peptides. An increase in protein sequence coverage is achieved by derivatization with these reagents or by combination of mass values before and after derivatization. Since a combination of 6-CH3-Nic-NHS and d3-labeled reagent (6-CD3-Nic-NHS) generates a 3 Da mass difference per reaction site, the d3-labeled reagent shifts the mass values of d0-labeled peptides according to the number of reactive amino groups in the peptides. In the case of tryptic peptides, the mass values of C-terminal arginine and lysine peptides are shifted by 3 and 6 Da, respectively. Further, the 3 Da mass difference between 6-CH3-Nic-NHS and 6-CD3-Nic-NHS offers a means for the relative quantification of protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Copyright © 2007 John Wiley & Sons, Ltd.

A sensitive method for the determination of polyamines in mammalian cells was described using electrospray ionization and time-of-flight mass spectrometer. This method was 50-fold more sensitive than the previous method using ionspray ionization and quadrupole mass spectrometer. The method employed the partial purification and derivatization of polyamines, but allowed a measurement of multiple samples which contained picomol amounts of polyamines. Time required for data acquisition of one sample was approximately 2 min. The method was successfully applied for the determination of reduced spermidine and spermine contents in cultured cells under the inhibition of aminopropyltransferases. In addition, a new proper internal standard was proposed for the tracer experiment using (15)N-labeled polyamines.