In laboratory experiments, many electrophilic cytotoxic agents induce cell death accompanied by reactive oxygen species (ROS) production and/or by glutathione (GSH) depletion. Not surprisingly, millimolar concentrations of N-acetylcysteine (NAC), which is used as a universal ROS scavenger and precursor of GSH biosynthesis, inhibit ROS production, restore GSH levels, and prevent cell death. The protective effect of NAC is generally used as corroborative evidence that cell death induced by a studied cytotoxic agent is mediated by an oxidative stress-related mechanism. However, any simple interpretation of the results of the protective effects of NAC may be misleading because it is unable to interact with superoxide (O2•-), the most important biologically relevant ROS, and is a very weak scavenger of H2O2. In addition, NAC is used in concentrations that are unnecessarily high to stimulate GSH synthesis. Unfortunately, the possibility that NAC as a nucleophile can directly interact with cytotoxic electrophiles to form non-cytotoxic NAC-electrophile adduct is rarely considered, although it is a well-known protective mechanism that is much more common than expected. Overall, apropos the possible mechanism of the cytoprotective effect of NAC in vitro, it is appropriate to investigate whether there is a direct interaction between NAC and the cytotoxic electrophile to form a non-cytotoxic NAC-electrophilic adduct(s).

• Klíčová slova
• N-acetylcysteine, N-acetylcysteine-electrophile adduct, electrophile, mechanism of protection, nucleophile,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

We studied the kinetics of the reaction of N-acetyl-l-cysteine (NAC or RSH) with cupric ions at an equimolar ratio of the reactants in aqueous acid solution (pH 1.4−2) using UV/Vis absorption and circular dichroism (CD) spectroscopies. Cu2+ showed a strong catalytic effect on the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical (ABTSr) consumption and autoxidation of NAC. Difference spectra revealed the formation of intermediates with absorption maxima at 233 and 302 nm (ε302/Cu > 8 × 103 M−1 cm−1) and two positive Cotton effects centered at 284 and 302 nm. These intermediates accumulate during the first, O2-independent, phase of the NAC autoxidation. The autocatalytic production of another chiral intermediate, characterized by two positive Cotton effects at 280 and 333 nm and an intense negative one at 305 nm, was observed in the second reaction phase. The intermediates are rapidly oxidized by added ABTSr; otherwise, they are stable for hours in the reaction solution, undergoing a slow pH- and O2-dependent photosensitive decay. The kinetic and spectral data are consistent with proposed structures of the intermediates as disulfide-bridged dicopper(I) complexes of types cis-/trans-CuI2(RS)2(RSSR) and CuI2(RSSR)2. The electronic transitions observed in the UV/Vis and CD spectra are tentatively attributed to Cu(I) → disulfide charge transfer with an interaction of the transition dipole moments (exciton coupling). The catalytic activity of the intermediates as potential O2 activators via Cu(II) peroxo-complexes is discussed. A mechanism for autocatalytic oxidation of Cu(I)−thiolates promoted by a growing electronically coupled −[CuI2(RSSR)]n− polymer is suggested. The obtained results are in line with other reported observations regarding copper-catalyzed autoxidation of thiols and provide new insight into these complicated, not yet fully understood systems. The proposed hypotheses point to the importance of the Cu(I)−disulfide interaction, which may have a profound impact on biological systems.

• Klíčová slova
• autoxidation, circular dichroism, copper catalysis, dicopper complex, dioxygen activation, disulfide, exciton coupling,
• MeSH
• acetylcystein * MeSH
• antioxidancia MeSH
• disulfidy MeSH
• kyslík chemie   MeSH
• měď chemie   MeSH
• oxidace-redukce MeSH
• reaktivní formy kyslíku MeSH
• sulfhydrylové sloučeniny * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcystein * MeSH
• antioxidancia MeSH
• disulfidy MeSH
• kyslík MeSH
• měď MeSH
• reaktivní formy kyslíku MeSH
• sulfhydrylové sloučeniny * MeSH

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

• Klíčová slova
• HL-60 cells, MPO, N-acetylcysteine, NOX, SOD, U937 cells, oxidative stress,
• MeSH
• acetylcystein farmakologie   MeSH
• HL-60 buňky MeSH
• katalasa genetika   MeSH
• leukemie farmakoterapie   genetika   metabolismus   MeSH
• lidé MeSH
• NADPH-oxidasa 2 genetika   MeSH
• oxidační stres účinky léků   MeSH
• peroxidasa genetika   MeSH
• proliferace buněk účinky léků   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• superoxiddismutasa genetika   MeSH
• U937 buňky MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcystein MeSH
• CYBB protein, human MeSH Prohlížeč
• katalasa MeSH
• MPO protein, human MeSH Prohlížeč
• NADPH-oxidasa 2 MeSH
• peroxidasa MeSH
• reaktivní formy kyslíku MeSH
• superoxiddismutasa MeSH