Oxidative Stress Induced by a Dihydropyrazine Derivative

Abstract

The Maillard reaction, which comprises a non-enzymatic reaction between reducing sugars and amino acids, produces hundreds of highly reactive compounds. Dihydropyrazine (DHP) is produced by condensation of 2 mol of D-glucosamine, 1) and some DHP derivatives generate various radical species that cause DNA strand cleavage in vitro.2) Since many pyrazine derivatives have been detected in various foods 3) and human urine, 4) we speculated that DHPs, which are presumed to be precursors of pyrazines, would be responsible for certain diseases. However, there are few reports regarding the biological and physiological roles of DHPs.In a previous paper, we reported that methyl-substituted dihydropyrazines (Me-DHPs), such as 2,3-dihydro-5,6-dimethylpyrazine, had DNA strand-breaking activity, inhibited growth and induced mutagenesis in Escherichia coli.5) Although phenyl-substituted dihydropyrazines (Ph-DHPs) have much higher DNA strand-breaking activities and produce higher levels of radical species than Me-DHPs, 6,7) DNA repair-deficient bacteria were less sensitive to Ph-DHPs than to Me-DHPs. 8) Recently, cyclohexyl-DHP, which was designed according to our hypothesis of a relationship between the chemical structures of DHP derivatives and DNA strandbreaking activities, 9) was shown to possess a stronger DNA strand-breaking activity than Me-DHPs and Ph-DHPs. 10)In the present study, we examined the cellular effects of a novel cyclohexyl-DHP with a cyclohexyl ring fused to the DHP. We found that it could strongly generate superoxide anions, and cause not only breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. MATERIALS AND METHODS Synthesis of DHPsA mixture of two isomers of cyclohexyl-DHP (Fig. 1), namely 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exotype), was prepared using a previously described method. 11) were obtained from the National BioResource Project (NIG, Japan). CATD [AB1157 katE katG] and SODD [AB1157 sodA sodB] were described in a previous paper. 8)DNA Strand-Breaking Assay Reaction mixtures (20 ml) containing 1 mg of plasmid pUC18 were incubated with DHP in the presence or absence of 1 mM CuCl 2 for 1 h at 37°C. The resulting mixtures were analyzed by 0.8% agarose gel electrophoresis and stained with 0.5 mg/ml ethidium bromide. The DNA bands were visualized with an Epi-LightUV FA500 (Taitec, Japan).Survival Assay Overnight cultures of E. coli were diluted by 100-fold with fresh LB medium and grown at 37°C until the OD 600 was approximately 0.1. Approximately 1ϫ10 8 bacteria were then challenged with DHP in the presence or absence of 1 mM CuCl 2 for 1 h with shaking at 37°C, plated on LB plates after serial dilutions in M9 salts and incubated overnight at 37°C. The survival rates were calculated as the percentages of cells surviving after challenge with DHP. All experiments were conducted at least four times. The Maillard reaction contributes to the complications of diabetes and normal aging. Dihydropyrazine...

Generation of Radical Species from Dihydropyrazines Having DNA Strand-Breakage Activity and Other Characteristics

“…The genotoxic 1,4,29) and cytotoxic 11,28,30) potentials of DHPs have been demonstrated previously. As stated earlier, the biological effects of DHPs are due to DHPs' dual capacity for high chemical reactivity and radical generation activity.…”

Glutathione depression by dihydropyrazine derivative

“…In addition, Cyclohexyl-DHP was shown to possess stronger DNA strand-breaking activity than Me-DHPs and PhDHPs (Maruoka et al, 2005;Yamaguchi et al, 2007). Moreover, we demonstrated that DHPs can potentially generate superoxide anions (Takechi et al, 2009), and not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Takechi et al, 2010).…”

Dihydropyrazine-Induced Inactivation of Glyceraldehyde-3-phosphate Dehydrogenase

“…11,14) Therefore, we examined and compared the generation of superoxide anions by three DHP derivatives, cyclohexyl-DHP, Me-DHP-1, and Ph-DHP-1, using the highly water-soluble tetrazolium salt WST-1.…”

“…14) In the present study, we examined enzyme inactivation by DHPs using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a convenient model protein, since GAPDH is known to be highly sensitive to oxidative damage. 15) Our observations reveal that DHPs inactivate GAPDH in vitro and in vivo.…”