Hans-Gert Korth

The pathobiochemistry of nitrogen dioxide.

Abstract Nitrogen dioxide (NO2) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed NO2. Peroxynitrite (ONOO/ONOOH) is believed to be an important endogenous source of NO2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of NO2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and βcarotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O2)dependent formation of peroxynitrate (O2NOO) and the central role of vitamin C are of special importance.

Hydrogen Peroxide Formation by Reaction of Peroxynitrite with HEPES and Related Tertiary Amines IMPLICATIONS FOR A GENERAL MECHANISM

Organic amine-based buffer compounds such as HEPES (Good’s buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimineN-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (⋅NO) and superoxide (O⨪2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mmSIN-1 was low (∼15 μm), incubation of SIN-1 with Good’s buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mm SIN-1 with 20 mm HEPES about 190 μm H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mm SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration ofp-hydroxyphenylacetic acid were strongly inhibited by 20 mm HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H2O2. Likewise, authentic peroxynitrite (1 mm) also induced the formation of both O⨪2 and H2O2 upon addition to HEPES (400 mm)-containing solutions in a pH (4.5–7.5)-dependent manner. In accordance with previous reports it was found that at pH ≥5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mm)-induced H2O2 formation (∼80 μm at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mm/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good’s buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate α-amino alkyl radicals with molecular oxygen leads to the formation of O⨪2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H2O2formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.

The Captodative Effect

Publisher Summary The concept of captodative substitution implies the simultaneous action of a captor (acceptor) and a donor substituent on a molecule. The chapter analyzes whether the claim of the proponents of the captodative effect has found experimental or theoretical support. The postulate of a synergetic action of a captor and a donor substituent at a radical centre and its chemical consequences has been discussed. Captodative substitution is that chemical consequences should be connected with this substitution pattern. For synthetic purposes, a captodative effect of a few kcal mol- might be helpful for the selection of a lower energy pathway in cases where a radical can react by several reaction paths, discriminated by activation barriers of closely similar energy. Synthetic applications have shown that captodative-substituted radicals generally behave like other stabilized, short-lived radicals. Left alone these radicals usually give dimers in high yield, as, for instance, allylic or benzylic radicals also do. As captodative substitution provides stability to a radical centre it is no surprise that captodative olefins are goodpartners in (2 + 2)-cycloadditions.

Advances in switchable supramolecular nanoassemblies.

Supramolecular nanoassemblies are gaining increasing importance as promising new materials with considerable potential for novel and promising applications. Within supramolecular nanoassemblies the connectivity of the monomeric units is based on reversible noncovalent interactions, like van der Waals interactions, hydrogen bonding, or ionic interactions. As the strength of these interactions depends on the molecular surrounding, the formation of nanoassemblies in principle can be controlled externally by changing the environment and/or the molecular shape of the underlying monomer. This way it is not only possible to switch the self-assembly on or off, but also to change between different aggregation states. In this minireview we present some recent selected approaches to supramolecular stimuli-responsive nanoassemblies.

Assessment of chelatable mitochondrial iron by using mitochondrion-selective fluorescent iron indicators with different iron-binding affinities.

Chelatable cellular iron, and chelatable mitochondrial iron in particular, has yet to be well characterized, so the overall strength with which these “loosely bound” iron ions (presumably mainly FeII) are intracellularly/intramitochondrially bound is unclear. We have previously reported the first selective mitochondrial iron indicator: rhodamine B 4‐[(1,10‐phenanthrolin‐5‐yl)aminocarbonyl]benzyl ester (RPA). With this compound as a model, we have now developed two additional mitochondrial iron indicators with very different iron‐binding affinities and have applied these to the study of the chelatable iron pool in the mitochondria of isolated rat liver cells. With the new indicator rhodamine B 4‐[(2,2′‐bipyridin‐4‐yl)aminocarbonyl]benzyl ester (RDA), with 2,2′‐bipyridine as chelating unit (log β3=17.5), essentially the same iron concentration (16.0±1.9 μM) was determined as with RPA (log β3=21.1), despite the four orders of magnitude difference in FeII‐binding affinity. This not only demonstrates the reliability of the procedure, but also confirms that iron complexation by these indicators does not induce any significant release of iron from the iron‐storage proteins on the timescale of the experiment. In contrast, the indicator rhodamine B 4‐[bis(pyridin‐2‐ylmethyl)aminomethyl]benzyl ester (PIRO), with an N,N‐bis(pyridin‐2‐ylmethyl)amine group as chelating component (log β2=12.2), could not compete against the array of endogenous ligands. The intramitochondrial concentrations of the three indicators were determined to be in the range of 100 μM: that is, about three orders of magnitude lower than the total concentration of endogenous compounds that might chelate iron ions. It is therefore estimated that chelatable mitochondrial iron ions are bound by endogenous ligands with apparent stability constants (log Kapp) of between 9 and 14.

Inhibition of peroxynitrite-induced nitration of tyrosine by glutathione in the presence of carbon dioxide through both radical repair and peroxynitrate formation.

Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2*) and trioxocarbonate(1-) (CO3*-) radicals. We have studied the mechanism by which glutathione (GSH) inhibits the NO2*/CO3*--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent tyrosine consumption (IC50 = 660 microM) as well as 3-nitrotyrosine formation (IC50) = 265 microM). From the determination of the level of oxygen produced or consumed under various initial conditions, it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS*) through reduction of dioxygen to superoxide (O2*-) and reaction with NO2* to form peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effective in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2* radicals is highly unlikely at physiological levels of GSH.

Cheletropic Traps for the Fluorescence Spectroscopic Detection of Nitric Oxide (Nitrogen Monoxide) in Biological Systems

Membrane-permeable, phenanthrene-derivedo-quinodimethanes of type 1 react with nitric oxide in a cheletropic fashion to produce fluorescent nitroxide radicals 2 and hydroxylamines 3. This method allows the sensitive and quantitative detection of nitric oxide production in biological samples by means of fluorescence microscopy, as is demonstrated by the monitoring of NO production from alveolar macrophages.

Cooperative self-assembly of discoid dimers: hierarchical formation of nanostructures with a pH switch.

Derivatives of the self-complementary 2-guanidiniocarbonyl pyrrole 5-carboxylate zwitterion (1) (previously reported by us to dimerize to 1•1 with an aggregation constant of ca. >10(10) M(-l) in DMSO) aggregate in a diverse manner depending on, e.g., variation of concentration or its protonation state. The mode of aggregation was analyzed by spectroscopic (NMR, UV) and microscopic (AFM, SEM, HIM, and TEM) methods. Aggregation of dimers of these zwitterions to higher supramolecular structures was achieved by introduction of sec-amide substituents at the 3-position, i.e., at the rearward periphery of the parent binding motif. A butyl amide substituent as in 2b enables the discoid dimers to further aggregate into one-dimensional (rod-like) stacks. Quantitative UV dilution studies showed that this aggregation is strongly cooperative following a nucleation elongation mechanism. The amide hydrogen seems to be essential for this rod-like aggregation, as neither 1 nor a corresponding tert-amide congener 2a form comparable structures. Therefore, a hydrogen bond-assisted π-π-interaction of the dimeric zwitterions is suggested to promote this aggregation mode, which is further affected by the nature of the amide substituent (e.g., steric demand), enabling the formation of bundles of strands or even two-dimensional sheets. By exploiting the zwitterionic nature of the aggregating discoid dimers, a reversible pH switch was realized: dimerization of all compounds is suppressed by protonation of the carboxylate moiety, converting the zwitterions into typical cationic amphiphiles. Accordingly, typical nanostructures like vesicles, tubes, and flat sheets are formed reversibly under acidic conditions, which reassemble into the original rod-like aggregates upon readjustment to neutral pH.

Involvement of Reactive Oxygen Species in the Preservation Injury to Cultured Liver Endothelial Cells

We have previously demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. In the present study, we report experimental evidence for the involvement of reactive oxygen species in this injury: LDH release during 48 h of cold incubation in UW solution was decreased from 40-55% under aerobic conditions to less than 20% under hypoxic conditions or by the presence of KCN (1 mM). Similar protection was achieved by the addition of the spin trap 5,5-dimethyl-1-pyrroline N-oxide, the hydroxyl radical scavenger dimethyl sulfoxide, or the flavonoid silibinin to UW solution under aerobic conditions. Preincubating the cells with the iron chelator deferoxamine even decreased the injury to less than 5%. The residual injury (as observed after longer incubation times) under hypoxic conditions or in cells preincubated with deferoxamine was no longer energy dependent. The amount of thiobarbituric acid-reactive substances markedly increased during cold incubation of the cells in UW solution. This increase was not observed in UW solution to which KCN had been added, i.e., under the conditions of energy depletion. These results suggest that an iron-dependent generation of reactive oxygen species with subsequent lipid peroxidation is involved in the pathogenesis of the injury to cultured liver endothelial cells in cold UW solution.

Nitric oxide detection and visualization in biological systems. Applications of the FNOCT method.

Abstract Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) were applied to specifically trap nitric oxide (NO) with high sensitivity. The fluorescent oquinoid ?electron system of the FNOCTs (? = 460 nm, ? = 600 nm) reacts rapidly with NO to a fluorescent phenanthrene system (? = 380 nm, ? = 460 nm). The cyclic nitroxides thus formed react further to nonradical products which exhibit identical fluorescence properties. Using the acid form of the trap (FNOCT-4), NO release by spermine NONOate and by lipopolysaccharide (LPS) activated alveolar macrophages were studied. A maximum extracellular release of NO of 37.5 nmol h[-1] (10[6] cells)[-1] from the macrophages was determined at 11 h after activation. Furthermore, intracellular NO release by LPSactivated macrophages and by microvascular omentum endothelial cells stimulated by the Ca[2+] ionophore A-23187, respectively, was monitored on the single cell level by means of fluorescence microscopy. After loading the cells with the membranepermeating acetoxymethylester derivative FNOCT-5,which is hydrolyzed to a nonpermeating dicarboxylate by intracellular hydrolases, NO formation by the endothelial cells started immediately upon stimulation, whereas start of NO production by the macrophages was delayed with a variation between 4 and 8 h for individual cells. These results demonstrate that the FNOCTs can be used to monitor NO release from single cells, as well as from NOdonating compounds, with high sensitivity and with temporal and spatial resolution.