Delivery and application of dietary polyphenols to target organs, tissues and intracellular organelles.

At present, dietary polyphenols are popular with consumers because regular consumption of polyphenol-rich foods is likely to be beneficial for human health. However, administrated polyphenols are extensively metabolized in the digestive tract or some other parts before reaching the target organs. Additionally, some of the polyphenols are photosensitive, easily oxidized and are in unfavorable forms. Therefore, a lot of work has been performed to ensure delivery of intact polyphenols to the target organs. We here summarize recent progress in polyphenol-delivery to individual organs, tissues, and cells, in regard to relatively new delivery systems. Polyphenol-delivery systems can be divided into three categories: (i) before delivery into the blood stream (skin, mouth, gastrointestine), (ii) in the blood stream (plasma), and (iii) after the blood stream (brain, spleen, bone marrow, kidney). Polyphenols before the delivery into blood stream must overcome several obstacles to avoid converting into inactive forms by commensal microorganisms, environmental pH, and some others. In the blood, plasma-polyphenol interactions and modifications are very effective for the bioavailability of polyphenols with numerous enzymes. Native forms of polyphenols, successfully out of the blood stream, further go through obstacles such as the blood brain barrier to reach target organs. Recent progress in delivering polyphenols is here discussed on 3 main delivery systems, nanoparticle, liposome, and microemulsion. Moreover, we also focused on delivery systems to intracellular organelles (cell surface, lysosome, mitochondria, nucleus), which are the final targets of polyphenols to perform their beneficial reactions.

The green tea polyphenol (-)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health.

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health.

Synthetic studies on novel 1,4-dihydro-2-methylthio-4,4,6-trisubstituted pyrimidine-5-carboxylic acid esters and their tautomers.

A mixture of alkyl 1,4-dihydro-2-methylthio-4,4,6-trisubstituted pyrimidine-5-carboxylate 1 and its tautomeric isomer, alkyl 1,6-dihydro-2-methylthio-4,6,6-trisubstituted pyrimidine-5-carboxylate 2 is synthesized by the Atwal-Biginelli cyclocondensation reaction of S-methylisothiourea hemisulfate salt 3 with 2-(gem-disubstituted)methylene-3-oxoesters 4 that can be accessed by the Lehnert procedure for the Knoevenagel-type condensation. The structures of the tautomeric products of the Atwal-Biginelli cyclocondensation reaction, 1 and 2, which are inseparable from each other, are determined unambiguously by (1)H-NMR spectroscopy at various temperatures and nuclear Overhauser enhancement spectroscopy (NOESY) experiment. Because these dihydropyrimidine products are otherwise inaccessible and thus hitherto unavailable, the synthetic methods established in this study will help to expand the molecular diversity of their related derivatives.

Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases.

vitamin D is 25-hydroxylated in the liver, before being activated by 1alpha-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min(-1)), vitamin D2 (0.16 min(-1)), and 1alpha-hydroxyvitamin D3 (2.2 min(-1)). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min(-1)) more efficiently than vitamin D2 (0.86 min(-1)). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2.

Studies on the transcriptional regulation of cholesterol 24-hydroxylase (CYP46A1): marked insensitivity toward different regulatory axes.

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.

Eight cytochrome P450s catalyze vitamin D metabolism.

Vitamin D3 plays a central role in calcium and phosphate homeostasis and is essential for the proper development and maintenance of bone. To exert its biological activities, vitamin D3 has to receive enzymatic transformation to the active form, 1,25-dihydroxyvitamin D3. The first step is the 25-hydroxylation reaction in the liver that produces 25-hydroxyvitamin D3, the major circulating form of vitamin D3. The 25-hydroxylation reaction is the prerequisite step for the subsequent 1 alpha-hydroxylation and 24-hydroxylation reactions in the kidney. The 1 alpha-hydroxylation reaction produces the active form of vitamin D3, whereas 24-hydroxylation reaction leads to inactivation. Both reactions are strictly controlled by parathyroid hormone, 1,25-dihydroxyvitamin D3, and calcium in a reciprocal manner in the kidney. At present, six cytochrome P450s (CYP2C11, 27A1, 2D25, 2R1, 3A4, and 2J3) are found to exhibit vitamin D 25-hydroxylation activities, and CYP27B1 and CYP24 are proved to be 1 alpha-hydroxylase and 24-hydroxylase, respectively. The main focus of this review is to summarize the properties of individual P450 in light of their catalytic activities to understand their physiological significance.

Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in gamma-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay.

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.

Identification of a novel rat microsomal vitamin D3 25-hydroxylase.

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.

Identification and characterization of mammalian 5-formyluracil-DNA glycosylase.

5-Formyluracil is a major oxidative thymine lesion with mutagenic and cytotoxic properties. In this study, we have partially purified and characterized a mammalian 5-formyluracil-DNA glycosylase (FDG) from rat liver. FDG was a monofunctional DNA glycosylase and removed 5-formyluracil, uracil, 5-hydroxyuracil, 5-hydroxylmethyluracil in single-stranded and double-stranded DNA. Several lines of evidence indicate that FDG is a rat SMUG1 homologue. Human SMUG1 also exhibited similar enzymatic properties.

Repair roles of hSMUG1 assessed by damage specificity and cellular activity.

Single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1) was previously identified as a putative backup enzyme of major mammalian uracil-DNA glycosylase (UDG). However, the subsequent studies have shown conflicting results about the substrate specificity of SMUG1. In the present study, to clarify the repair role of SMUG1, we determined the damage specificity of purified human SMUG1 (hSMUG1) and its contribution to repair of oxidized bases in HeLa cell extracts.

Repair of oxidative cytosine damage by DNA glycosylases.

5-Hydroxyuracil (HOU) and 5-hydroxycytosine (HOC) are major oxidative lesions of cytosine with mutagenic potentials. Therefore, HOU and HOC need to be removed from DNA to avoid mutation. In this study, oligonucleotide substrates containing HOU and HOC were synthesized by DNA polymerase reactions and tested for DNA glycosylases. Ung exhibited an extremely low activity for HOU as compared to uracil (U). In contrast, hSMUG1 excised HOU and U with a comparable efficiency. Ung and hSMUG1 did not excise HOC.

Mammalian 5-formyluracil-DNA glycosylase. 1. Identification and characterization of a novel activity that releases 5-formyluracil from DNA.

5-Formyluracil (fU) is a major oxidative thymine lesion produced by reactive oxygen species and exhibits genotoxic and cytotoxic effects via several mechanisms. In the present study, we have searched for and characterized mammalian fU-DNA glycosylase (FDG) using two approaches. In the first approach, the FDG activity was examined using purified base excision repair enzymes. Human and mouse endonuclease III homologues (NTH1) showed a very weak FDG activity, but the parameter analysis and NaBH(4) trapping assays of the Schiff base intermediate revealed that NTH1 was kinetically incompetent for repair of fU. In the second approach, FDG was partially purified (160-fold) from rat liver. The enzyme was a monofunctional DNA glycosylase and recognized fU in single-stranded (ss) and double-stranded (ds) DNA. The most purified FDG fraction also exhibited monofunctional DNA glycosylase activities for uracil (U), 5-hydroxyuracil (hoU), and 5-hydroxymethyluracil (hmU) in ssDNA and dsDNA. The fU-excising activity of FDG was competitively inhibited by dsDNA containing U.G, hoU.G, and hmU.A but not by intact dsDNA containing T.A. Furthermore, the activities of FDG for fU, hmU, hoU, and U in ssDNA and dsDNA were neutralized by the antibody raised against SMUG1 uracil-DNA glycosylase, showing that FDG is a rat homologue of SMUG1.

Mammalian 5-formyluracil-DNA glycosylase. 2. Role of SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil and other oxidized and deaminated base lesions.

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.

DNA-protein cross-link formation mediated by oxanine. A novel genotoxic mechanism of nitric oxide-induced DNA damage.

Chronic inflammation is a risk factor for many human cancers, and nitric oxide (NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. In this report, we report a novel genotoxic mechanism of NO that involves DNA-protein cross-links (DPCs) induced by oxanine (Oxa), a major NO-induced guanine lesion. When a duplex DNA containing Oxa at the site-specific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between Oxa and protein. The rate of DPC formation with DNA glycosylases was approximately two orders of magnitude higher than that with histone and HMG protein. Analysis of the reactivity of individual amino acids to Oxa suggested that DPC formation occurred between Oxa and side chains of lysine or arginine in the protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing Oxa. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis.

Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid.

Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (V(max)/K(m)) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [(3)H]Xan from the substrate. The treatment of E.coli with N-methyl-N'-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA(+) but not alkA(-) strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan.

Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: translesion DNA synthesis, nucleotide insertion, and extension kinetics.

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3'-5' exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f(ins) = V(max)/K(m) for insertion) and extension (f(ext) = V(max)/K(m) for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f(ins) = 1) dTMP (2.4 x 10(-4)) approximately dAMP (8.1 x 10(-5)) > dGMP (4.5 x 10(-7))), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f(ext) = 1) > mFapyG:T (4.6 x 10(-3)) mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.

Detection of NO-induced DNA lesions by the modified aldehyde reactive probe (ARP) assay.

When DNA is exposed to NO or HNO2, oxanine (Oxa) is formed as a major guanine lesion. For highly sensitive detection of Oxa using ARP, a probe molecule for DNA damage detection, the reactivity of ARP to Oxa was examined. Oxa site-specifically embedded in an oligonucleotide reacted with ARP but it took relatively long time (ca. 24 h) to completely convert Oxa to an ARP-labeled form.

Extensive methylation of CpG island of CYP24 gene in osteoblastic ROS17/2.8 cells.

CYP24 is a target gene of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and is induced in the cells expressing vitamin D receptor (VDR) in response to 1,25-(OH)2D3. The osteoblastic ROS17/2.8 cells abundantly express VDR and have been used for the promoter analysis of many of vitamin D3 target genes. However, unlike other cells, ROS cells did not induce CYP24 expression upon 1,25-(OH)2D3 treatment. It has been reported that the methylation of CpG islands of a promoter region is involved in gene silencing. Methylation analysis of the CYP24 gene revealed that the CpG island in 5' part of the transcription unit is extensively methylated. This result suggests that unresponsiveness of the CYP24 gene to 1,25-(OH)2D3 would result from the methylation of the promoter region.