3-Hydro-2,2,5,6-tetramethylpyrazine: A novel inducer of zinc transporter-1 in HepG2 human hepatocellular carcinoma cells.

Dihydropyrazine compounds, including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are low-molecular-weight glycation products spontaneously generated in vivo and also ingested via food. Our preliminary study using microarray analysis demonstrated that DHP-3 induced zinc transporter-1 (ZnT-1) in HepG2 cells. It is well known that the increase of intracellular zinc is a sensitive stimulating factor for ZnT-1 protein induction; however, there is little information about the induction of ZnT-1 by low-molecular-weight chemical compounds. Here, we attempted to clarify the mechanism of ZnT-1 induction by DHP-3. A significant increase of ZnT-1 mRNA was observed 6h after DHP-3 treatment at concentrations over 0.5mM, and disappeared 24h after exposure. This induction pattern followed that of metal-responsive transcription factor 1 (MTF-1) mRNA, a metalloregulatory protein that serves as a major transcription factor of ZnT-1. Moreover, DHP-3 yielded transcriptional activation of MTF-1 in a luciferase reporter assay. The intracellular zinc content was unaffected by the compound; however, oxidative stress was observed in cells under the same conditions that activated the MTF-1 signaling pathway. These results suggest that DHP-3 is a novel ZnT-1 inducer and acts via activation of the MTF-1 signaling pathway. Additionally, the activation of MTF-1 by this compound likely occurs through oxidative stress.

Identification of dihydropyrazine-glutathione adducts.

Dihydropyrazines (DHPs) are glycation intermediates generated both in vivo and in food. DHPs can lead to the formation of a variety of different radical species, which can lead to DNA damage and enzyme inhibition. In addition, the presence of DHPs can lead to a decrease in cellular glutathione (GSH) levels, and induce the expression of antioxidant genes. In this study, the products resulting from the reaction of DHP with GSH have been analyzed in detail, with some of the products being separated by reversed-phase HPLC. The structures of the isolated DHP-GSH adducts were determined by FAB-MS and NMR analyses. These data suggested that the reaction of DHP with a thiol moiety could be involved in oxidative stress, because an increase in the amount of DHP-GSH adducts would result in a decrease in the cellular GSH levels.

Effect of dihydropyrazines on human hepatoma HepG2 cells: a comparative study using 2,3-dihydro-5,6-dimethylpyrazine and 3-hydro-2,2,5,6-tetramethylpyrazine.

Dihydropyrazines (DHPs) are glycation products that are nonenzymatically generated in vivo and in food. In this study, we compared the effects of 2,3-dihydro-5,6-dimethylpyrazine (DHP-1), a low toxicity DHP, and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), a high toxicity DHP on the redox indices in HepG2 cells. An apparent increase in intracellular hydrogen peroxide concentration was observed at 24 hr after 1 mM DHP-3 treatment. In addition, DHP-3 exposure significantly increased the mRNA levels of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC), which are stress-responsive genes, at 6 hr (HO-1 and GCLC), 12 hr (HO-1 and GCLC) and 24 hr (GCLC) after exposure. These indices, with the exception of the increase in GCLC mRNA after a 6 hr exposure, were not affected by treatment with 1 mM DHP-1. HO-1, GCLC, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels also increased at 6 hr (Nrf2), 12 hr (Nrf2, HO-1 and GCLC) and 24 hr (GCLC) after DHP-3 treatment. The increase in HO-1 and Nrf2 protein levels were observed with lower concentration (0.5 mM) of DHP-3, and in agreement with this, antioxidant responsive element-luciferase reporter activity was significantly increased with exposure to at least 0.5 mM DHP-3. These results support our previous report establishing that oxidative stress is in part involved in the effects of DHP on mammalian cells. Additionally, our results suggest that the cell response to DHP-3 exposure was exerted via the activation of the Nrf2-ARE signal pathway.

Selenium-binding protein 1: its physiological function, dependence on aryl hydrocarbon receptors, and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

BACKGROUND: Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1. METHODS: To address this issue, we generated Selenbp1-null [Selenbp1 (-/-)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model. RESULTS: Selenbp1 (-/-) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (-/-) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (-/-) and (+/+) mice. CONCLUSIONS: The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity. GENERAL SIGNIFICANCE: Selenbp1 (-/-) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.

Possible involvement of glutathione balance disruption in dihydropyrazine-induced cytotoxicity on human hepatoma HepG2 cells.

Dihydropyrazines (DHPs), formed by nonenzymatic glycation, are known to exert various effects in vitro and in vivo, such as generation of radical species, DNA strand breakage, enzyme inhibition, and inhibition of bacterial growth. However, their effects on mammalian cells remain elusive. To address this issue, we investigated the effects of a range of DHP concentrations on human hepatoma HepG2 cells using 2,3-dihydro-5,6-dimethylpyrazine (DHP-1), 2,3-dihydro-2,5,6-trimethylpyrazine (DHP-2), and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3) as model compounds. All of the tested compounds exerted cytotoxic activity against HepG2 cells in the range of 10 µM-1 mM, and significantly so at the highest concentration. DHP-3 was the most effective drug, and it also caused a significant decrease in the ratio of intracellular reduced and oxidized glutathione (GSH/GSSG). In addition, the cytotoxic effect of DHP-3, but not DHP-1 and DHP-2, was enhanced by the inhibition of GSH biosynthesis using 100 µM l-buthionine-(S,R)-sulfoximine (BSO). From these results, it is suggested that the mechanisms of cytotoxicity exerted by DHP-3 are distinct from those exerted DHP-1 and DHP-2. In addition, it is possible that the disruption of intracellular glutathione balance induced by DHP-3 is related to its effect on HepG2 cells.

Generation of radical species from dihydropyrazines having DNA strand-breakage activity and other characteristics.

The various biological activity of dihydropyrazines(DHPs)due to the radical generation potency has been described in previous papers. Detailed data about radical species generating be mentioned here. The electron spin resonance (ESR) spin-trapping technique revealed that DHPs generate free radical species such as ·OH, ·OOH, ·CHR(2) and ·CR(3). Oxygen radicals and two carbon-centered radicals were detected as adducts of the spin traps DMPO and DBNBS, respectively. All the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)- and 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS)-adducts of compounds DHP-1-8 exhibited approximately the same signal patterns, with various levels of intensity depending on the substituent of the dihydropyrazine ring. The ESR signal intensity of DHPs also increased remarkably upon addition of Cu(2+), resulting that the effects of DHPs were enhanced.

2,3,7,8-tetrachlorodibenzo-p-dioxin potentially attenuates the gene expression of pituitary gonadotropin ß-subunits in a fetal age-specific fashion: a comparative study using cultured pituitaries.

Our previous studies have demonstrated that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a reduction in gonadotropin biosynthesis in the fetal pituitary, resulting in the attenuated expression of steroidogenic proteins in the fetal gonads and the impairment of sexual behaviors in adulthood. However, the mechanism of the attenuation remains unknown. To address this issue, we investigated whether TCDD affects the pituitary production of gonadotropins, using cultured pituitary. In the absence of gonadotropin-releasing hormone (GnRH), a regulator of gonadotropin biosynthesis, TCDD did not affect the expression of gonadotropin mRNAs both in fetal and postnatal pituitaries. On the other hand, in the presence of GnRH, TCDD interfered with the synthesis of gonadotropin ß-subunit mRNAs only in the fetal pituitary. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and a PKA activator (8-bromoadenosine-3' 5'-cyclic monophosphate) induced the expression of gonadotropin mRNAs in the fetal pituitary. Among the subunits, only the induction of ß-subunit was reduced by TCDD treatment. These results suggest that TCDD reduces gonadotropin biosynthesis via damage to GnRH-stimulated PKC and PKA signaling in a ß-subunit- and fetal age-specific manner.

Glutathione depression by dihydropyrazine derivative.

Dihydropyrazine (DHP), which is formed by nonenzymatic glycation, generates various radical species that lead to DNA damage and enzyme inhibition. In this study, we examined the reaction between DHP derivatives and glutathione (GSH). DHP exposure caused more intense growth inhibition of a GSH-deficient mutant Escherichia coli strain compared with the wild-type strain. DHP-exposed mouse fibroblasts showed a decrease in the cellular GSH level. The obtained data suggested that the reaction of DHP with GSH possibly potentiates cellular stress via the depletion of cellular GSH levels.

Latent enamine functionality of 5-methyl-2,3-dihydropyrazines.

Tautomerization of methyl-substituted dihydropyrazine (DHP) derivatives to their latent enamine form was investigated theoretically and empirically. Among two types of hydrogen transfer model simulated by means of density functional theory calculation, a simple intramolecular hydrogen shift mechanism for 5,6-dimethyl-2,3-dihydropyrazine (1) and 5-methyl-6-phenyl-2,3-dihydropyrazine (3) required high activation energies for tautomerism, while a water-assisted intermolecular hydrogen transfer mechanism gave smaller activation energies (about 160 kJ/mol). Examination of the deuterium exchange reaction of 3 in 50% (v/v) D(2)O/dimethyl sulfoxide-d(6) solution revealed temperature-dependent and stepwise deuterium exchange of the 5-methyl group. Reaction of compound 3 with phenyl isocyanate in acetonitrile afforded a mono adduct (7) at the 5-methyl group, and a cyclic adduct (8). These results represent evidence of tautomerism of 5-methyl-2,3-dihydropyrazines (imine forms) to the latent enamine tautomers, and suggest that DHPs may behave as enamines to a significant degree under physiological conditions.

Phenyl-substituted dihydropyrazines with DNA strand-breakage activity.

Monophenyl-substituted dihydropyrazines (Ph-DHP-1 to 4) of 2,3-dihydro-5,6-dimethylpyrazine (Me-DHP-1), which have the inductive effects of apoptosis and mutagenesis, were synthesized and their biological effect was investigated in terms of DNA strand-breakage. Differences between the phenyl- and methyl-substituted dihydropyrazines were examined.

Dihydropyrazine-induced inactivation of glyceraldehyde-3-phosphate dehydrogenase.

Dihydropyrazine (DHP), which is produced during the Maillard reaction, generates radicals that not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. In the present study, we show that three DHP derivatives, which generated superoxide anions, caused inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SH-compounds, such as cysteine, dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, and N-acetyl-cysteine, suppressed the inhibition of GAPDH by DHP in vitro, although the effect of DHP on GAPDH was not reversed by DTT. In addition, DHP-exposed Escherichia coli showed almost unaffected growth on plates containing a rich medium, but poor growth on plates containing M9 synthetic medium with glucose as the sole carbon source. Furthermore, DHP-exposed E. coli exhibited reduced GAPDH activity. These findings indicate that DHP disturbs the glycolytic pathway by inhibiting GAPDH activity.

Tetraazaindenes derived from dihydropyrazines with DNA strand-breaking activity.

Dihydropyrazines (DHPs), which exhibit DNA strand-breaking activity and other biological activities associated with the generation of radical species, reacted with thiourea to give tetraazaindene (TAI) derivatives, despite thiourea is a well known radical scavenger. The structural determination of the TAIs was carried out and formation mechanism of TAI was investigated.

Chemical reactivity of dihydropyrazine derivatives. Cycloaddition behavior toward ketenes.

The cycloaddition behavior of dihydropyrazines toward ketenes was investigated using single-crystal X-ray structures of the cycloadducts and density functional theory (DFT) calculation data. The reaction proceeds via a stepwise pathway involving an orientation complex prior to formation of the betaine intermediate. This is followed by electrocyclization to afford the 1 : 1 and 1 : 2 adducts bearing beta-lactam ring(s).

Oxidative stress induced by a dihydropyrazine derivative.

The Maillard reaction contributes to the complications of diabetes and normal aging. Dihydropyrazines (DHPs), which are produced during the Maillard reaction, generate radicals and possess DNA strand-cleaving activities in vitro. In the present study, we evaluated the genotoxic and cytotoxic potentials of a DHP derivative, cyclohexyl-DHP, which is obtained as a mixture of two isomers, 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exo-type), fused with a cyclohexyl ring. Cyclohexyl-DHP caused DNA strand breaks in plasmid pUC18, especially in the presence of Cu(2+). By using Escherichia coli mutant strains, we observed that cyclohexyl-DHP exposure strongly reduced the survival rate of a cytosolic sodium dodecyl sulfate (SOD)-deficient strain (sodA sodB), significantly reduced the survival rates of DNA repair-deficient strains (recA and uvrB) and mildly reduced the survival rate of a catalase-deficient strain (katE katG) compared with the survival rate of the wild-type strain. Addition of Cu(2+) enhanced the cell killing ability of cyclohexyl-DHP. The frequency of mutations induced by cyclohexyl-DHP increased dose-dependently in the sodA sodB strain. Assays with the highly water-soluble tetrazolium salt WST-1 revealed that cyclohexyl-DHP strongly generated superoxide anions. Moreover, cyclohexyl-DHP elevated the protein carbonyl levels in E. coli. These findings indicate that cyclohexyl-DHP could potentially generate superoxide anions, and cause not only breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins.

Effects of phenyl derivatives of dihydropyrazines with ability to generate radical species on Escherichia coli.

Phenyl-substituted dihydropyrazines (Ph-DHPs) are derivatives of 2,3-dihydro-5,6-dimethylpyrazine (Me-DHP). Upon the addition of Cu(2+), Me-DHP inhibits the growth of Escherichia coli by generating hydroxyl and carbon-centered radicals that cause DNA strand breakage. Here, we investigated the toxic effect of Ph-DHPs in several DNA repair-deficient or detoxifying enzyme-deficient mutant strains. Ph-DHPs caused cytotoxic and genotoxic damage, but, in a sodA sodB strain, the effects in the presence or absence of Cu(2+) were different than those of Me-DHP. Our results suggest that the action of the generated superoxide anion in the interior side of the cell is remarkable.

The relationship between the chemical structures of dihydropyrazine derivatives and DNA strand-breakage activity.

Dihydropyrazine, a compound derived from sugars, possesses DNA strand-breakage activity. The relationship between the activity as assayed using pBR 322 ccc-DNA and the chemical structures of derivatives of dihydropyrazine (DHPs) has been investigated. The addition of Cu(2+) enhanced the activity remarkably. The introduction of a methyl or phenyl group onto the DHP ring or a cyclohexyl group fused onto the DHP ring also increased the activity. These properties indicated that the activity was due to the facility of electron release from the DHP ring, followed by radical generation. The determination of ionization potential and electrostatic potential values, and bond dissociation energy via semi-empirical MO calculations suggested strongly that the activity is induced by a DHP ring structure that contains a configuration suitable for hyperconjugation.

Mutation spectrum induced by dihydropyrazines in Escherichia coli.

Dihydropyrazine (DHP), which induces mutagenesis in E. coli, was investigated. From analyzing mutations in the chromosomal rpoB gene, the mutation spectrum in uvrB strain revealed the different behavior on exposure to two DHP derivatives 3-hydro-2,2,5,6-tetramethylpyrazine (HTMP), and 2,3-dihydro-5,6-dimethylpyrazine (DHDMP). A higher level of DHP-induced mutation was observed, with base substitutions at G : C pairs predominant. HTMP and DHDMP increased the frequency of G : C to T : A transversions. HTMP increased the frequency of G : C to A : T transitions, than did DHDMP. These findings suggest that DHPs prefer to attack the G : C pair and that different DHP derivatives may prefer distinct mutagenic base pairs; and further, that nucleotide excision repair may be involved in the repair of DHP-induced mutations.

Synthesis of new dihydropyrazines with DNA strand-breakage activity.

Treatment of 1,2-cyclohexanedione with 1,2-diamines, e.g. ethylenediamine and cis-(and trans-)1,2-diaminocyclohexane, caused [4+2] cyclocondensation to give the corresponding dihydropyrazine derivatives (compounds 1-6). They exhibited stronger DNA strand-breakage activity than that of dihydropyrazines, which has already been reported in previous papers.

The Role of dihydropyrazines in accelerated death of Escherichia coli on addition of copper(II).

Dihydropyrazines (DHPs), which are derived from sugars, inhibit the growth of Escherichia coli. Addition of copper(II) ion (Cu2+) accelerates the effect of DHPs, resulting in cell death. Investigation of the lethal effect in several DNA repair-deficient or detoxifying enzyme-deficient mutant strains and radical scavengers suggested that the cytotoxic and genotoxic damage was caused by radical species (hydroxyl, superoxide anion, and carbon-centered radicals) generated from reaction of DHPs with Cu2+.

Cloning, sequence analysis, and expression of the gene encoding Sphingomonas paucimobilis FP2001 alpha-L -rhamnosidase.

The gene (rhaM) encoding the alpha-L-rhamnosidase of Sphingomonas paucimobilis FP2001 was cloned, sequenced, and expressed in Escherichia coli. The rhaM consisted of 3,354 nucleotides and had a promoter and Shine-Dalgarno sequences typical in bacteria. The rhaM encoding a protein (Rham) deducted from the sequence consisted of 1,117 amino acids and had a putative signal peptide of 25 amino acids. Rham has no similarity to other known rhamnosidases. Rham has a sugar-binding domain of glycoside hydrolase family 2, which has been well conserved in beta-glucuronidase, beta-mannosidase, and beta-galactosidase, in its C-terminal region. Rham is possibly a member of a new bacterial subfamily in glycoside hydrolase family 78 (alpha-L-rhamnosidase). RT-PCR analysis of rhaM mRNA indicated that the induction of alpha-L-rhamnosidase by the addition of L-rhamnose occurred on the transcriptional level.