Radical scavenging and chemical repair of rutin observed by pulse radiolysis: as a basis for radiation protection

Abstract

Pulse radiolysis was conducted to investigate: several fundamental reactions of a natural flavonoid, rutin, and its glycosylated form (αG-rutin) as a basis for their radiation protection properties; the reactions with ^?OH (radical scavenging) and dGMP radical, dGMP^? (chemical repair), which was used as a model of initial and not yet stabilised damage on DNA. Three absorption peaks were commonly seen in the reactions of the flavonoids with ^?OH, showing that their reactive site is the common structure, i.e. aglycone. One among the three peaks was attributed to the flavonoid radical produced as a result of the removal of a hydrogen atom. The same peak was found in their reactions with dGMP^?, showing that dGMP^? is chemically repaired by obtaining a hydrogen atom supplied from the flavonoids. Such a spectral change due to the chemical repair was as clear as never reported. The rate constants of the chemical repair reaction were estimated as (9?±?2)×10⁸ M^?1 s^?1 and (6?±?1)×10⁸ M^?1 s^?1 for rutin and αG-rutin, respectively. The rate constants of the radical scavenging reactions towards ^?OH were estimated as (1.3?±?0.3)×10¹⁰ M^?1 s^?1 and (1.0?±?0.1)×10¹⁰ M^?1 s^?1 for rutin and αG-rutin, respectively. In addition, there was no obvious difference between rutin and αG-rutin, indicating that the glycosylation does not change early chemical reactions of rutin.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (^?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against ^?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H²O²), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a ^?OH-trapping agent after UVB exposure (305?nm) to H²O² for 1?min in the presence of rebamipide. The signal intensity of ^?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10¹⁰, 8.16?×?10⁹ and 1.65?×?10¹⁰?M^-1?s^-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the ^?OH generated by UVB/H²O². Specific formation of this product explained the molecular characteristics of rebamipide as a potential ^?OH scavenger.     

Antioxidative iridoid glycosides from the sky flower (Duranta repens Linn)

Phytochemical investigations were performed on the EtOAc-soluble fraction of the whole plant of the sky flower (Duranta repens) which led to the isolation of the iridoid glycosides 1–6. Their structures were elucidated by both 1D and 2D NMR spectroscopic analysis. All the compounds showed potent antioxidative scavenging activity in four different tests, with half maximal inhibitory concentration (IC₅₀) values in the range 0.481–0.719?mM against DPPH radicals, 4.07–17.21 µM for the hydroxyl radical (^?OH) inhibitory activity test, 43.3–97.37 µM in the total reactive oxygen species (ROS) inhibitory activity test, and 3.39–18.94 µM in the peroxynitrite (ONOO^?) scavenging activity test. Duranterectoside A (1) displayed the strongest scavenging potential with IC₅₀ values of (0.481?±?0.06?mM, 4.07?±?0.03, 43.30?±?0.05, 3.39?±?0.02?µM) for the DPPH radicals, ^?OH inhibitory activity test, total ROS inhibitory activity test and the ONOO^? scavenging activity test, respectively.     

The role of CS<Subscript>2</Subscript> in CS<Subscript>2</Subscript>/NMP mixed solvent in weakening the hydrogen bond of OH–N in coal: a DFT investigation

The interaction processes of trace amounts of N-methyl-2-pyrrolidinone (NMP), CS₂/NMP (1:1 by volume) and pure NMP solvent with the hydrogen bond of OH?N in coal were constructed and simulated by density functional theory methods. The distances and bond orders between the main related atoms, and the hydrogen bond energy of OH?N were calculated. The calculated results show that pure NMP solvent does not weaken the hydrogen bond of OH?N in coal. However, trace amounts of NMP and CS₂/NMP (1:1 by volume) have a strong capacity to weaken the hydrogen bond of OH?N in coal. The H2–N3 distances are elongated from 1.87 Å to 3.80 Å and 3.44 Å, the bond orders of H2–N3 all disappear, and the corresponding hydrogen bond energies of OH?N in coal decrease from 45.72 kJ mol^?1 to 7.06 and 11.24 kJ mol^?1, respectively. These results show that CS₂ added to pure NMP solvent plays an important role in releasing the original capacity of NMP to weaken the hydrogen bond of OH?N in coal, in agreement with experimental observations.     

Measurement of OH radical CT for inactivating Cryptosporidium parvum using photo/ferrioxalate and photo/TiO2 systems

Aim: This study investigates the inactivation of Cryptosporidium parvum using the OH radical and reports the OH radical CT (OH radical concentration × contact time) values for C. parvum inactivation. Methods and Results: Although a wealth of information has demonstrated the efficacy of the microbial inactivation activity of the OH radical, no studies have performed a quantitative estimation of the OH radical for C. parvum inactivation. The CT value of the OH radical required for 2 log C. parvum inactivation was measured with two OH radical‐generating systems, photo/ferrioxalate and photo/TiO₂. The OH radical was approx. 10⁴–10⁷‐fold more effective for microbial inactivation than other popular chemical disinfectants such as ozone, chlorine dioxide and free chlorine. Conclusions: The OH radical appears to be suitable for microbial inactivation with a calculated CT value required for 2 log C. parvum inactivation of 9·3 × 10^?5 mg min l^?1. Significance and Impact of the Study: This study is the first report of an investigation on the role of the OH radical in the photo/ferrioxalate and photo/TiO₂ systems and on the OH radical CT required for C. parvum inactivation.     

Radical scavenging ability of some compounds isolated from Piper cubeba towards free radicals

The purpose of this study was to identify the antioxidant activity of 16 compounds isolated from Piper cubeba (CNCs) through the extent of their capacities to scavenge free radicals, hydroxyl radical (HO^?), superoxide anion radical () and 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH^?), in different systems. Electron paramagnetic resonance (EPR) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide, DMPO, as the spin trap, and chemiluminescence techniques were applied. Using the Fenton‐like reaction [Fe(II) + H₂O₂], CNCs were found to inhibit DMPO? OH radical formation ranging from 5 to 57% at 1.25 mmol L^?1 concentration. The examined CNCs also showed a high DPPH antiradical activity (ranging from 15 to 99% at 5 mmol L^?1 concentration). Furthermore, the results indicated that seven of the 16 tested compounds may catalyse the conversion of superoxide radicals generated in the potassium superoxide/18‐crown‐6 ether system, thus showing superoxide dismutase‐like activity. The data obtained suggest that radical scavenging properties of CNCs might have potential application in many plant medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Indigo stability: an ab initio study

This work shows that indigo's high stability can be attributed both to the large π conjugation inside the molecule and to intra- and intermolecular hydrogen bonds. The theoretical investigation of indigo's electronic structure has been performed using high-level methods. To understand the interactions in solid state, calculations of the dimer system with both molecules in the same plane was carried out. In the monomer, two intramolecular hydrogen bridges between amino and carbonyl groups occupy positions that would otherwise be the most reactive ones for nucleophilic and electrophilic attacks. In the dimer, amino and carbonyl groups on different monomers form intermolecular multicentred non-linear hydrogen bonds in six-member rings, protecting again the same reactive centres and explaining the limited solubility of indigo. The addition of the free radical OH breaks the central C = C double bond, the conjugation and the hydrogen bridges as a first step. The Gibbs energy calculation favours the addition of OH radical over C1.     

Mechanism for the depolymerization of cellulose under alkaline conditions

The mechanism for the hydroxyl-radical-induced depolymerization of cellulose under alkaline conditions in air was investigated using density functional theory at the B3LYP/6-31+G(d,p) level as well as electron transfer theory. The pathway for the depolymerization of cellulose was obtained theoretically and H abstraction from the C(3) atom of the pyran ring during the cleavage of the glucosidic bond was found to be the rate-limiting step due to its high energy barrier (16.81 kcal/mol) and low reaction rate constant (4.623?×?10⁴ mol L^?1 s^?1). Calculations of the electron transfer between O₂ and the saccharide radical performed with the HARLEM software package revealed that following the H abstraction, the oxygen molecule approaches C(2) on the saccharide radical and obtains an electron from the radical, even though no bond forms between the oxygen molecule and the radical. The rate constant for electron transfer could be as high as 1.572?×?10¹¹ s^?1. Furthermore, an enol intermediate is obtained during the final stage of the depolymerization.     

Biotransformation of plantain pseudostem fibres using local enzyme sources; analysis of their potential as commercial poultry feed

Plantain pseudo-stem fibres (PPS) were valorized in this study by subjecting to simultaneous saccharification and fermentation (SSF) and afterwards, their complex carbohydrates and monosaccharides, mycotoxins, protein qualities, and free radical scavenging potentials were compared to those of commercial poultry feeds (CPF). The SSF of PPS was achieved using digestive juice of the snail; Archachatina marginata, and yeast, while standard methods like HPLC-UV, HPLC-DAD, monosaccharides and mycotoxin kits, and UV-VIS spectrophotometry were used for analysis. The cellulose, hemicellulose, pectin, lignin, extractives, and acetyl contents of PPS were significantly (p?<?.05) reduced when subjected to SSF. Glucose (41.1%), galactose (11.2%), mannose (1.7%), and fucose (1.8%) contents of the SSF-PPS were higher than those of the PPS and CPF while CPF showed higher contents of arabinose (8.2%), fructose (18.3%), and rhamnose (1.7%). No mycotoxin was detected in the PPS, while all aflatoxins (B₁, B₂, G₁, and G₂), citrinin, fumonisin B₁, and B₂, ochratoxin A and B contents of SSF-PPS were equivalent to those for CPF. Patulin (5.52×10^?4?µg/kg) and zearalenone (7.76×10^?6?µg/kg) contents of the SSF-PPS were lower than those for CPF (1.50×10^?3?µg/kg and 1.13×10^?5?µg/kg respectively). The total amino acids (TAA), total non-essential and essential amino acids (TNEAA and TEAA), total basic and branched chain amino acids (TBAA and TBCAA) of the SSF-PPS were higher than those of the PPS and CPF while the free radical scavenging potentials of the SSF-PPS were mostly concentration dependent, and showed significantly higher ABTS, DPPH, Ferric, OH, lipid peroxide, and superoxide radical scavenging potentials than the standards used. This study has shown that the valorization of the agricultural residue using SSF, improves carbohydrate, protein, mycotoxins, and in-vitro antioxidant properties suitable enough for poultry feeding.     

Different mechanisms of hydroxyl radical production susceptible to purine P2 receptor antagonists between carbon monoxide poisoning and exogenous ATP in rat striatum

Abstract

Previous studies have suggested that carbon monoxide (CO) poisoning stimulates cAMP production via purine P2Y11-like receptors in the rat striatum, activating cAMP signaling pathways, resulting in hydroxyl radical (^?OH) production. Extracellular ATP was thought likely to trigger the cascade, but the present study has failed to demonstrate a clear increase in the extracellular ATP due to CO poisoning. The CO-induced ^?OH production was attenuated by the P2Y11 receptor antagonist NF157, in parallel with its abilities to suppress the CO-induced cAMP production. The ^?OH production was more strongly suppressed by a non-selective P2 receptor antagonist, PPADS, which had no effect on cAMP production. More selective antagonists toward the respective P2 receptors susceptible to PPADS, including NF279, had little or no effect on the CO-induced ^?OH production. The intrastriatal administration of exogenous ATP dose-dependently stimulated ^?OH production, which was dose-dependently antagonized by PPADS and NF279 but not by NF157. Exogenous GTP and CTP dose-dependently stimulated ^?OH production, though less potently. The GTP-induced ^?OH production was susceptible to both of NF279 and PPADS, but the CTP-induced ^?OH production was resistant to PPADS. The mechanism of ^?OH production may differ between CO poisoning and exogenous ATP, while multiple P2 receptors could participate in ^?OH production. The CO-induced ^?OH production was susceptible to the inhibition of NADPH oxidase, but not xanthine oxidase. Also, the NADPH oxidase inhibition suppressed ^?OH production induced by forskolin, a stimulator of intracellular cAMP formation. It is likely that ^?OH is produced by NADPH oxidase activation via cAMP signaling pathways during CO poisoning.     

Activity and function in lung injuries due to sulphur mustard

Aim. Pulmonary complications are known to occur in over half the patients exposed to sulphur mustard. Many studies have focused on the clinical complications, often ignoring the pathogenesis of sulphur mustard. Also, the reasons for the variable severity of lung injuries caused by sulphur mustard are unclear. Hence, the current study was performed to evaluate the correlation between superoxide dismutase (SOD) and catalase (CAT) activity and pulmonary function in patients exposed to sulphur mustard. Methods. Our study was a comparative cross-sectional survey. Two hundred and fifty incident survivors were selected from the Sardasht population who were exposed to sulphur mustard in 1987. A control group from non-exposed civilians was also selected. We used a pulmonary function test, and SOD and CAT activity was measured in these groups. Results. The mean SOD activity in the healthy control group (70.5±10.8 U ml^?1) was higher than in the moderate-to-severe group (67.0±6.1 U ml^?1) (p <0.001, one-tail ANOVA, least significant difference (LSD) post hoc). The mean activity in the mild group (72.5±6.9 U ml^?1) was no higher than in the healthy control group (70.5±10.8 U ml^?1) (p=0.095 one-tail ANOVA, LSD post hoc). The mean CAT activity in the healthy control group (4.9±1.5 U ml^?1) was lower than in the moderate-to-severe group (8.0±1.8 U ml^?1) (p <0.001, one-tail ANOVA, LSD post hoc) and in the mild group (7.5±1.5 U ml^?1) (p=0.012 one-tail ANOVA, LSD post hoc). Conclusion. According to our findings, it is reasonable to hypothesize that re-establishment of the activation–inactivation or oxidant–antioxidant balance in favour of the activation and antioxidant balances would be useful as a therapeutic strategy to suppress pathological mechanisms underlying lung injuries.     

Dominance of a 675?nm chlorophyll(ide) form upon selective 632.8 or 654?nm laser illumination after partial protochlorophyllide phototransformation

The phototransformation pathways of protochlorophyllide forms were studied in 8?C14-day-old leaves of dark-germinated wheat (Triticum aestivum L.) using white, 632.8?nm He?CNe laser and 654?nm laser diode light. The photon flux density (PFD) values (0.75?C360???mol photons?m^?2?s^?1), the illumination periods (20?ms?C10?s) and the temperature of the leaves (between ?60?°C and room temperature) were varied. The 77?K fluorescence spectra of partially phototransformed leaves showed gradual accumulation or even the dominance of the 675?nm emitting chlorophyllide or chlorophyll form at room temperature with 632.8?nm of PFD less than 200???mol photons?m^?2?s^?1 or with 654?nm of low PFD (7.5???mol photons?m^?2?s^?1) up to 1?s. Longer wavelength (685 or 690?nm) emitting chlorophyllide forms appeared at illuminations under ?25?°C with both laser lights or at room temperature when the PFD values were higher or the illumination period was longer than above. We concluded that the formation of the 675?nm emitting chlorophyllide form does not indicate the direct photoactivity of the 633?nm emitting protochlorophyllide form; it can derive from 644 and 657?nm forms via instantaneous disaggregation of the newly-produced chlorophyllide complexes. The disaggregation is strongly influenced by the molecular environment and the localization of the complex.     

Antioxidant phenylpropanoid glycosides from Buddleja davidii

Phytochemical investigations on the n-BuOH-soluble fraction of the whole plant of Buddleja davidii led to the isolation of the phenylpropanoid glycosides 1-10. Their structures were determined by 1D and 2D NMR spectroscopic techniques. All the compounds showed potent antioxidative activity in three different tests, with IC₅₀ values in the range 4.15-9.47 μM in the hydroxyl radical (˙OH) inhibitory activity test, 40.32-81.15 μM in the total ROS (reactive oxygen species) inhibitory activity test, and 2.26-7.79 μM in the peroxynitrite (ONOO^?) scavenging activity test. Calceolarioside A (1) displayed the strongest scavenging potential with IC₅₀ values of (4.15?±?0.07, 40.32?± 0.09, 2.26?±?0.03μM) for ˙OH, total ROS and scavenging of ONOO^?, respectively.     

Synthesis,antioxidant and radical scavenging activities of novel benzimidazoles

The synthesis and antioxidant evaluation of some novel benzimidazole derivatives (10–24) are described. Antioxidant properties of the compounds were investigated employing various in vitro systems viz., microsomal NADPH-dependent inhibition of lipid peroxidation (LP), interaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and scavenging of superoxide anion radical. Compounds 12 and 13 showed very good antioxidant capacity and were 17–18 -fold more potent than BHT (IC₅₀ 2.3 × 10^{? 4}M) with 1.3 × 10^{? 5}M and 1.2 × 10^{? 5}M IC₅₀ values, respectively, by interaction of the stable DPPH free radical.     

Oxygen radical induced alterations in polyclonal IgG

In the sera and synovial fluid of patients with rheumatoid arthritis, part of the IgG fraction is found in an aggregated and fluorescent form. Oxygen-free radicals have been implicated in this denaturation, although the precise radical species responsible is unknown. In this work, oxygen-free radicals generated radiolytically were allowed to attack polyclonal IgG in solution. OH radicals induced aggregation of the monomer and a new fluorescence appeared in the visible region (Ex 360 nm, Em 454 nm). The superoxide radical anion was found to be inert in both these respects, whilst peroxy radicals induced autofluorescence without concomitant aggregation. The results suggest that OH.and/or peroxy radical attack may be an in vivo mechanism for IgG denaturation.     

Gas-phase reaction of benzo[a]anthracene with hydroxyl radical in the atmosphere: products,oxidation mechanism,and kinetics

Polycyclic aromatic hydrocarbons (PAHs) have induced large-scale and long-term environmental contamination due to heavy emissions, toxicity, and persistence. The investigation of the ultimate sink of PAHs in the atmosphere is very important. In this work, using quantum chemistry methods, the reaction mechanism of hydroxyl radical-initiated oxidation of benzo[a]anthracene (BaA) in the atmosphere was studied. The products resulted from the gas-phase reaction of BaA with hydroxyl radical include benzo[a]anthracenols, dialdehydes, ketones, epoxides, etc. Applying Rice-Ramsperger-Kassel-Marcus (RRKM) theory, the overall rate constant for reactions of ?OH addition to BaA was estimated to be 4.82?×?10^?11 cm³ molecule^?1 s^?1 at 298 K and 1 atm. The lifetime of BaA in the atmosphere with respect to hydroxyl radical was calculated to be 5.92 h.     

Luminescence of bacteriorhodopsin from Halobacterium halobium and its connection with the photochemical conversions of the chromophore

Red luminescence of purple membranes from Halobacterium halobium cells in suspension, dry film or freeze-dried preparations was studied and its emission, excitation and polarization spectra are reported. The emission spectra have three bands at 665–670, 720–730 and at 780–790 nm. The position (maximum at 580 nm) and shape of the excitation spectra are close to those of the absorption spectra. The spectra depend on experimental conditions, in particular on pH of the medium. Acidification increases the long wavelength part of the emission spectra and shifts the main excitation maximum 50–60 nm to the longer wavelength side. Low-temperature light-induced changes of the absorption, emission and excitation spectra are presented. Several absorbing and emitting species of bacteriorhodopsin are responsible for the observed spectral changes. The bacteriorhodopsin photoconversion rate constant was estimated to be about 1 · 10¹¹ s^?1 at ? 196°C from the quantum yields of the luminescence (1 · 10^?3) and photoreaction (1 · 10^?1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. High degree of luminescence polarization (about 45–47%) throughout the absorption and fluorescence spectra and its temperature independence show that there is no energy transfer between bacteriorhodopsin molecules and no chromophore rotation during the excitation lifetime. In carotenoid-containing membranes, energy migration from the bulk of carotenoids to bacteriorhodopsin was not found either. Bacteriorhodopsin phosphorescence was not observed in the 500–1100 nm region and the emission is believed to be fluorescence by nature.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.