Sara Goldstein

The chemistry and biological activities of N-acetylcysteine

BACKGROUND N-acetylcysteine (NAC) has been in clinical practice for several decades. It has been used as a mucolytic agent and for the treatment of numerous disorders including paracetamol intoxication, doxorubicin cardiotoxicity, ischemia-reperfusion cardiac injury, acute respiratory distress syndrome, bronchitis, chemotherapy-induced toxicity, HIV/AIDS, heavy metal toxicity and psychiatric disorders. SCOPE OF REVIEW The mechanisms underlying the therapeutic and clinical applications of NAC are complex and still unclear. The present review is focused on the chemistry of NAC and its interactions and functions at the organ, tissue and cellular levels in an attempt to bridge the gap between its recognized biological activities and chemistry. MAJOR CONCLUSIONS The antioxidative activity of NAC as of other thiols can be attributed to its fast reactions with OH, NO2, CO3(-) and thiyl radicals as well as to restitution of impaired targets in vital cellular components. NAC reacts relatively slowly with superoxide, hydrogen-peroxide and peroxynitrite, which cast some doubt on the importance of these reactions under physiological conditions. The uniqueness of NAC is most probably due to efficient reduction of disulfide bonds in proteins thus altering their structures and disrupting their ligand bonding, competition with larger reducing molecules in sterically less accessible spaces, and serving as a precursor of cysteine for GSH synthesis. GENERAL SIGNIFICANCE The outlined reactions only partially explain the diverse biological effects of NAC, and further studies are required for determining its ability to cross the cell membrane and the blood-brain barrier as well as elucidating its reactions with components of cell signaling pathways.

The reaction of NO· with O2·− and HO2·−: A pulse radiolysis study

The reactions of NO. with O2.- and with HO2. were studied using the pulse radiolysis technique under pseudo first order conditions where ([O2.-]o + [HO2.]o) > [NO.]o at pH 3.3-10.0. The rate constant of the reaction of NO. with O2.- was determined both by monitoring the decay of O2.- at 250 nm and the formation of ONOO- at 302 nm to be (4.3 +/- 0.5) x 10(9) M-1s-1, independent of ionic strength and pH in the range of 6.1-10.0. The rate constant of the reaction of NO. with HO2.- was determined by following the decay of HO2. at 250 nm to be (3.2 +/- 0.3) x 10(9) M-1s-1 at pH 3.3.

The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from these systems from the toxicity of O2

Abstract Cooper complexes of 1,10-phenanthroline and some substituted 1,10-phenanthroline cleave DNA in the presence of a reducing agent and molecular oxygen. Generally, the damage is attributed to hydroxyl radicals which are formed through the Haber-Weiss reaction. It is assumed that this reaction occurs with the ternary metal complexes with the biological target and the mechanism is defined as the “site specific mechanism.” In these systems, O2− drives the cycle through the reduction of copper(II). On the other hand, these same copper complexes catalyze the dismutation of O2− and thus should protect the systems from O2− toxicity. In this article, the toxicity of these complexes is explained on kinetic grounds. A general discussion on the various factors which could cause the metal ions or their complexes to act either as protectors from O2− toxicity or as sensitizers of toxic effects of O2− is given.

Reactions of PTIO and carboxy-PTIO with *NO, *NO2, and O2-*.

Nitronyl nitroxides, such as derivatives of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIOs), react with ·NO to form the corresponding imino nitroxides (PTIs) and ·NO2. PTIOs are considered as monitors of ·NO, stoichiometric sources of ·NO2, biochemical and physiological effectors, specific tools for the elimination of ·NO, and potential therapeutic agents. However, a better understanding of the chemical properties of PTIOs, especially following their reaction with ·NO, is necessary to resolve many of the reported discrepancies surrounding the effects of PTIOs and to better characterize their potential therapeutic activity. We have generated electrochemically the oxidized and reduced forms of PTIO and carboxy-PTIO (C-PTIO), characterized their absorption spectra, and determined the reduction potentials for the oxoammonium/nitroxide and nitroxide/hydroxylamine couples. The rate constants for the reaction of ·NO2 with PTIO and C-PTIO to form the corresponding oxoammonium cations (PTIO+s) and nitrite were determined to be (1.5 - 2) × 107 m-1 s-1. We have also shown that the reactions of PTIO+s with ·NO form PTIOs and NO2-. The rate constants for these reactions are approximately 30-fold higher than those for the reactions of PTIOs with ·NO or \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{.}}}\) \end{document}. The present results show that (i) the reaction of PTIOs with ·NO forms solely PTIs and NO2- where [NO2-]/[PTI] varies between 1 and 2 depending on the steady-state concentrations of ·NO. Consequently, quantitation of ·NO is valid only at sufficiently low fluxes of ·NO; (ii) the reaction of PTIOs with ·NO can be used as a valid source of ·NO2 only when the latter is effectively scavenged by an appropriate reductant; and (iii) the formation of peroxynitrite cannot be efficiently inhibited by PTIOs even under relatively low fluxes of ·NO and O2 and millimolar levels of PTIOs.

Mannitol as an OH scavenger in aqueous solutions and in biological systems

By using the technique of pulse radiolysis to generate OH. radicals, we have determined through the competition with SCN-, I- and Fe(CN)4-(6) the rate constant of mannitol with OH. radical at pH = 7 to be (1.8 +/- 0.4) X 10(9)M-1 s-1.

The chemistry of peroxynitrite: implications for biological activity.

In biological systems, nitric oxide (NO) combines rapidly with superoxide (O2-) to form peroxynitrite ion (ONOO-), a substance that has been implicated as a culprit in many diseases. Peroxynitrite ion is essentially stable, but its protonated form (ONOOH, pKa = 6.5 to 6.8) decomposes rapidly via homolysis of the O-O bond to form about 28% free NO2 and OH radicals. At physiological pH and in the presence of large amounts of bicarbonate, ONOO- reacts with CO2 to produce about 33% NO2 and carbonate ion radicals (CO3-) in the bulk of the solution. The quantitative role of OH/CO3(-) and NO2 radicals during the decomposition of peroxynitrite (ONOOH/ONOO-) under physiological conditions is described in detail. Specifically, the effect of the peroxynitrite dosage rate on the yield and distribution of the final products is demonstrated. By way of an example, the detailed mechanism of nitration of tyrosine, a vital aromatic amino acid, is delineated, showing the difference in the nitration yield between the addition of authentic peroxynitrite and its continuous generation by NO and O2- radicals.

Direct and indirect oxidations by peroxynitrite, neither involving the hydroxyl radical.

A new mechanism (Mechanism III) that combines features of mechanisms suggested earlier (Goldstein and Czapski, Inorg. Chem. 34:4041-4048; 1995; Pryor, Jin, and Squadrito Proc. Natl. Acad. Sci. USA 91:11173-11177; 1994) is proposed for oxidations by peroxynitrite. In Mechanism III, oxidations by peroxynitrite can take place either directly by ground-state peroxynitrous acid, ONOOH, or indirectly by ONOOH*, where ONOOH* is an activated form of peroxynitrous acid. In the direct oxidation pathway the reaction is first order in peroxynitrite and first order in substrate, and the oxidation yield approaches 100%. In the indirect oxidation pathway the reaction is first order in peroxynitrite and zero order in substrate. In the presence of sufficient concentrations of a substrate that reacts by the indirect oxidation pathway, about 50-60% of the ONOOH directly isomerizes to nitric acid, and about 40-50% of the ONOOH is converted into ONOOH*. Thus, the oxidation yields by the indirect pathway will not exceed 40-50%, and there will always be a residual yield of nitrate even in the presence of very high concentrations of the substrate. Competitive inhibition studies with various free radical scavengers showed that in some cases these scavengers have no effect on oxidation yields. In others, only partial inhibition was observed, far less than that predicted from to the known rate constants for the reactions of these scavengers with the hydroxyl radical. There are some cases where the extent of inhibition correlates well with the known rate constants of the reactions of these scavengers with hydroxyl radical; nevertheless, even in these cases, the involvement of hydroxyl radicals in indirect oxidations by peroxynitrite is ruled our on the basis of kinetics and oxidation yields. Thus, direct oxidations by peroxynitrite are explained in terms of ONOOH, and indirect oxidations in terms of ONOOH*, and substrates can react by one or both of these pathways.

Mechanisms of the reactions of some copper complexes in the presence of DNA with superoxide, hydrogen peroxide, and molecular oxygen.

The kinetics and the reaction mechanism of some copper complexes of l,IO-phenanthroline, 5-nitrc1, IO-phenanthroline, and 2,2’-bipyridine with 02-, Hz02, and O2 in the presence of calf thymus DNA have been investigated with use of the pulse radiolysis technique. We have found that both copper(I1) and copper(1) complexes bind to DNA. The ternary complexes react very slowly with 02relative to the free complexes, while the rates of the oxidation of free and bound cuprous complexes by H202 are almost the same. Therefore these ternary copper complexes turned out to be good catalysts of the reaction between 02and Hz02. A complex between the chelating agent 1,lO-phenanthroline (OP) and copper(I1) is able to induce the degradation of DNA in the presence of a reducing agent.’-’ N o primary sequence specificity is apparent in the scission reaction* which proceeds under a variety of experimental conditions. These include incubtation of DNA, OP, and copper(I1) ions with the following: (a) reducing agents such as thiol or ascorbate in the presence of molecular oxygen;’” (b) systems generating the superoxide radical in the presence of molecular o ~ y g e n ; ~ . ~ (c) NADH and hydrogen pero~ide;~.’ (d) reducing agents and hydrogen p e r o ~ i d e . ~ The degradation of DNA was inhibited by intercalating agents and any reagent which reduced the concentration of either the cuprous complex (e.g., neocupr~ine)*-~g~ or hydrogen peroxide (e.g., ~a ta lase) .~” The sensitivity of the reaction to other inhibitors depended on the pathway for the generation of the cuprous complex and hydrogen peroxide (e.g., superoxide dismutase (SOD) inhibited the reaction potentiated by NADH and hydrogen peroxide but had no effect where thiol and hydrogen peroxide were p r e ~ e n t . ~ ~ 5-NO2-OP and 5-CLOP were more effective than OP in cleaving DNA while 5-CH3-OP was less effective than OP under comparable conditions.’,’ The cuprous complex of 2,2’-bipyridine (bpy) was unable to degrade DNA at similar concentrations used for OP,3-5 although the coordination chemistry, the kinetics, and the mechanism of the oxidation of this complex by oxygen and hydrogen peroxide are similar to that of OP.93’0 Moreover, it is known that complexes of bpy as well as those of OP bind to The reaction mechanism for this process has not yet been determined. It is believed that the cuprous complex intercalates with DNA and that the subsequent oxidation by hydrogen peroxide causes the damage due to the formation of OH. at the binding site.4” The binding constants of the various copper complexes to DNA and the kinetics and mechanism of the oxidation of the ternary complexes by oxygen, hydrogen peroxide, and superoxide radicals have not yet been determined. The understanding of the kinetics and mechanism of these reactions may shed light on the mechDNA.” ( I ) DAurora, V.; Stern, A. M.; Sigman, D. S. BBRC 1977, 78, 170. (2) Sigman, D. S.; Graham, D. R.; DAurora, V.; Stern, A. M. J . Biol. (3) Doweny, K. M.; Que, B. G.; So, A. G. BBRC 1980, 93, 264. (4) Que, B. G.; Doweny, K. M.; So, A. G. Biochemistry 1980, 19, 5987. (5) Marshall, L. E.; Graham, D. R.; Reich, K. A.; Sigman, D. S . Bio(6) Gutteridge, J. M.; Halliwell, B. Biochem. Phormacol. 1982. 31, 2801. (7) Reich, K. A,; Marshall, L. E.; Graham, D. R.; Sigman, D. S. J . Am. (8) Pope, L. M.; Reich, K. A,; Graham, D. R.; Sigman, D. S . J . Biol. (9) Goldstein, S.; Czapski, G. J . Am. Chem. Sot . 1983, 105, 7276. (IO) Goldstein, S . ; Czapski, G. Inorg. Chem. 1985, 24, 1087. ( 1 I ) Howe-Grant, M.; Lippard, S . J. Biochemisfry 1979, 18, 5762. Chem. 1979, 254, 12269. chemistry 1981, 20, 244. Chem. SOC. 1981, 103, 3582. Chem. 1982, 257, 12121. anism of DNA cleavage initiated by the various copper complexes. Experimental Section Materials. All chemicals employed were of analytical grade and were used as received: calf thymus DNA, type I, 2,2’-bipyridine, and sodium formate (Sigma Chemical Co.), l,lO-phenanthroline, 5-nitrophenanthroline (Fluka), H202 (Merck), SOD (Diagnostic Data Int.), cupric sulfate, monosodium and disodium phosphate (Mallinckrodt). All solutions were prepared in distilled water which was further purified by a Millipore reagent grade water system. A stock solution of DNA was prepared as 1 mg/mL containing 1 mM sodium phosphate buffer at pH 7. The concentration of DNA per nucleic acid phosphate was determined spectrophotometrically at 260 nm with z = 6875 M-’ cm-‘.I2 The cuprous complexes were generated by using the pulse radiolysis technique in oxygenated solutions containing 0.02 M sodium formate and 1 mM sodium phosphate buffer at pH 7. Under these conditions all the radicals formed by irradiation reduce the cupric c~mplexes . ’~’~ Kinetic studies were followed at 435 nm, where the various cuprous complexes a b s ~ r b . ~ . ’ ~ The concentration of H202 was determined with ferrous ~ u l f a t e . ’ ~ Apparatm. UV-visible absorption spectra were recorded with a Bauch and Lomb Model Spectronic 2000 spectrophotometer. The pulse radiolysis setup consisted of a Varian 7715 linear accelerator. The pulse duration ranged from 0.1 to 1.5 p s with a 200 mA current of 5 MeV electrons. The total concentration of the various cuprous complexes produced per pulse (1-15 pM) was evaluated with the use of a (0P),Cu2+ dosimeter. The yield of (OP)2Cu+ in oxygenated formate solution was assumed to be G = 6.05 and c = 6770 M-’ cm-’ at 435 Irradiation was carried out in a 2 cm long optical spectrosil cell with use of three light passes. A 150-W xenon lamp was used as the analytical light source and appropriate light filters were used to avoid photochemistry and to eliminate any scattered light. The detection system included a grating monochromator and an IP28 photomultiplier. The signal was transferred to a Nova 1200 minicomputer via either a Biomation 8100 or an analog-to-digital converter. The analysis of the data was carried out with the Nova 1200 minicomputer. Results and Discussion A. ”be Reduction of Copper(I1) by 0, in the Presence of DNA. In the irradiation of aqueous solutions containing formate ions and oxygen, the superoxide radical is p r ~ d u c e d . ’ ~ . ’ ~ As the pK of H02 is 4.8,I4J6 the reducing radical is mainly 02at pH 7. When the cupric complexes of OP, 5-N02-OP, or bpy (CuL?’) are present in excess relative to [02-],, reaction 1 takes place: ki CUL22+ + 0 2 CUL2+ + 0 2 (1) (12) Felsenfeld, G.; Hirschman, S. Z . J . Mol. Biol. 1965, 13, 407. (13) Matheson, M. S.; Dorfman, L. M. “Pulse Radiolysis”; MIT Press: (14) Bielski, B. H. J. Photochem. Photobiol. 1978, 28, 645. (15) Holm, N. W.; Berry, R. J. “Manual on Radiation Chemistry“; Marcel (16) Behar, D.; Czapski, G.; Rabani, J . ; Dorfman, L. M.; Schwartz, H. Cambridge, MA, 1969. Dekker Inc.: New York, 1970; pp 313-317. A. J. Phys. Chem. 1970, 74, 3209. 0002-7863/86/ 1508-2244

Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite.

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Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms

The measured parameters for the formation of peroxynitrous acid via the reaction of acidified hydrogen peroxide with nitrous acid and its self-decomposition corroborate with an earlier suggested mechanism in which H2NO2+ nitrosates H2O2. The activation energies for the formation and decay of peroxynitrous acid have been determined to be 15 and 19 kcal/mol, respectively. We found that perchlorate, nitrate, sulfate and phosphate ions have no effect on the formation and decay rates, whereas chloride ions enhance the rate of the formation of peroxynitrous acid at low peroxide concentrations, and have no effect at high peroxide concentrations. This suggests that at relatively low concentration of H2O2, Cl- competes with H2O2 for H2NO+ to yield NOCl, which may also nitrosate H2O2. Simulation of the experimentally observed parameters for the decay and formation rates suggests that it is not possible to obtain 100% yield of peroxynitrite under any condition. High yields of peroxynitrite were obtained at room temperature using an efficient double mixer where acidified peroxide was mixed with nitrite; after an appropriate delay, the reaction was quenched with strong alkali. An excess of more than 10% of H2O2 over nitrite, or vice versa, is sufficient to get ca. 85-90% of peroxynitrite, almost free from nitrite or H2O2, respectively. The results also suggest that conventional use of ice-cold solutions of the reactants and the alkali solutions is not required if an efficient mixer and appropriate quenching times are available.