The Role of Dihydropyrazines in Accelerated Death of Escherichia coli on Addition of Copper(II)

Abstract

In a recent paper, 2) we demonstrated the biological effects, such as growth inhibition and mutagenesis in Escherichia coli, of dihydropyrazines (DHPs). The DHPs revealed single strand-cleavage 3) on the covalently closed circular DNA of plasmid pBR322, especially in the presence of Cu 2ϩ . Since the participation of Cu 2ϩ enhanced the cleaving activity, it is likely that the effects of DHPs on E. coli also would be influenced. Here, we examined the effects of DHPs on E. coli upon the addition of Cu 2ϩ . Pyrazine derivatives have been detected in the urine of humans with diabetes mellitus, 4) which suggests that DHPs, which are precursors of pyrazines, might be involved in diabetes mellitus, although this remains unconfirmed. DHPs also are produced from the accumulation of the heme precursor, 5-aminolevulinic acid (ALA), in lead poisoning and some types of porphyries. 5,6) We have reported that DHPs induce apoptosis of cultured cells, 7) and, therefore investigated other potential biological effects caused by DHPs. The sugar origin of DHPs and their prevalence in the human body suggests that these compounds may be involved in various internal injuries in vivo. 7) MATERIALS AND METHODS Synthesis of DHPsThe DHPs (Fig. 1) were synthesized by condensation of diketones and diamines. 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) were prepared by the method of Yamaguchi et al. 3)Bacterial Strains E. coli K-12 strains used include the following. AB1157 [ara-14 argE3 galK2 his-4 lacY1 leuB6 mtl-1 proA2 rpsL31 supE44 thi- Survival Assay E. coli cultured overnight were diluted 100-fold in fresh LB medium and grown at 37°C until the OD 600 was approximately 0.1. Cells were challenged with DHPs for 1 h, incubated with shaking at 37°C, plated on LB agar, and incubated overnight at 37°C. Survival rates were calculated as the percentage of cells surviving after challenge by DHP-1, DHP-2 or DHP-3. All experiments were conducted at least four times.Enzymes and Chemicals Bovine erythrocyte superoxide dismutase (5000 units/mg), bovine liver catalase (11500 units/mg), dimethyl sulfoxide (DMSO), and 2-mercaptoethylamine hydrochloride (MEA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). RESULTSThe effect of DHPs in the presence of Cu 2ϩ was examined through the measurement of the E. coli viability. A wild-type strain, recA, and uvrB strain were incubated with or without Cu 2ϩ using three DHP types as shown in Fig. 1, which revealed in vitro DNA strand-cleaving activity of the order DHP-3ϾDHP-2ϾDHP-1.3) As shown in Fig. 2, the effect of DHPs in all strains of E. coli was accelerated greatly by addition of Cu 2ϩ . This is definitive evidence that the recA and uvrB strains possess greater sensitivity to DHPs upon addition of Cu 2ϩ compared to the wild-type strain. The effect in the recA strain was obvious, even without Cu 2ϩ . Therefore, this effect on E. coli must involve specific DNA damage caused by radical species generat...

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

“…Hence, it is predictable that DHPs cleave DNA strands by a) The spin amounts (µM) of DMPO-adducts of hydroxyl and carbon-centered radicals were estimated from the ESR spectra of DHPs (1.5 mM) in 50 mM Tris-HCl buffer containing 10% DMSO at 30 min after mixing, by using of a standard solution (2 µM) of 2,2,6,6-tetramethyl-4-piperidinol-1-oxyl. DHP-4 exhibits a remarkable effect 27) on E. coli in vivo due to the generation of a perhydroxyl radical species. It had been described due to catalase inhibition effect that H 2 O 2 in the reduction pathway from ·O 2 − to ·OH, may be concerned in same way in the reaction.…”

Tetraazaindenes Derived from Dihydropyrazines with DNA Strand-Breaking Activity

“…As thiourea served not as a radical scavenger but as a reactant with DHPs, the reaction afforded TAIs as products. According to the similar methods in our previous papers, [17][18][19] the growth inhibition and lethal effect of TAIs on E. coli were confirmed based on experiments using certain combinations (TAI only, TAIϩCu ϩthiourea. TAIs exhibited effects almost the same as those of DHPs, the lethal effect being somewhat stronger than that of DHP at 20 mM.…”

Oxidative Stress Induced by a Dihydropyrazine Derivative

“…The recA and uvrB strains were highly sensitive to Me-DHPs 20,21) and weakly sensitive to Ph-DHPs. 8) In addition, the uvrB strain showed an increased mutation frequency.…”

“…20,22) By contrast, the sodA sodB strain was highly sensitive to Ph-DHPs 8) and weakly sensitive to Me-DHPs. 20,21) Thus, Me-DHPs exert genotoxic effects while Ph-DHPs exert cytotoxic effects. Addition of Cu 2ϩ , which is expected to promote a Fenton-type reaction, 23) enhanced both the DNA strand-breaking and bactericidal activities of DHPs.…”