Thiol repair

Hydrogen-atom donation from thiols to carbon-centered radicals was too often assumed to be the only property of thiols that is important in free radical processes in biology. Moreover, it was a common presumption that reaction (1) was the end of the biological pathway in which thiols repair radicals. Equilibria turned out to be much more important in sulfur radical chemistry than was first thought. For instance, the hydrogen-donation reaction was found to be a reversible equilibrium over 30 years after it was first observed. ... 

Knowledge of the interaction of thiols with DNA-derived radicals is much less extensive. Moreover, one function of endogenous thiols is to repair radical damage in nucleic acids. Surprisingly, little was known about the rate constants for the thiol-repair reactions with pyrimidine-derived radicals having well-defined structures (Pyr ) [reaction (2)] ... 

One possibihty for minimizing oxidized protein damage is the thiol repair (Fig. 3). This repair system requires either glutathione or the thioredoxin system. The thioredoxin/thioredoxin reductase repair system [10] is able to reduce disulfide bonds. It can dethiolate protein disulfides and thus is an extremely important regulator for redox homeostasis in the cells. Thioredoxin is a smaU ubiquitous protein that contains a pair of cysteines that undergo reversible oxidation and are re-reduced by the enzyme thioredoxine reductase. The thioredoxin reductase transfers electrons from NADPH to thioredoxin via a flavin. 

Besides thiol repair there also exists direct repair for one of the oxidation products of methionine, methionine sulfoxide. The enzyme peptide methionine sulfoxide reductase reduces the methionine sulfoxide formed in proteins due to oxidation and is therefore able to reconstitute the normal protein (Fig. 3). Besides methionine sulfoxide there exists a further oxidation product of methionine, methionine sulfone, which can not be repaired. The cycle of methionine oxidation and efficient methionine sulfoxide repair, and the early and easy oxidation of the methionine in proteins, led some authors to hypothesize that methionine acts as an intramolecular antioxidant for some proteins and so protects other amino acids from oxidation [12]. Besides the peptide methionine sulfoxide reductases, there also exists methionine reductases able to... 

While the chief theory for the oxygen effect is considered to be the fixation model (oxygen reacts with the DNA damage site and fixes the damage before thiol repair), however, the oxygen effect may also involve the formation of sulfoxyl radicals [110]. In this model, repair proceeds by reactions such as reactions (36) and (37). With oxygen present then a series of reactions (38)-(41) take place ... 

The outcome of rapid radiation chemical processes in mammalian cells is to cause a variety of longer-Hved physical alterations in the DNA. Of these, double-strand breaks (DSBs) appear to be most frequently involved in cell killing if not correctly repaired. In general, thiols protect against DSB induction in proportion to their effect on cell killing (7), although there are exceptions (8). 

The Michael reaction involves conjugate addition of a nucleophile onto an a,P-unsaturated carbonyl compound, or similar system. Such reactions take place in nature as well, and some can be potentially dangerous to us. For example, the a,P-unsaturated ester ethyl acrylate is a cancer suspect agent. This electrophile can react with biological nucleophiles and, in so doing, bind irreversibly to the nucleophile, rendering it unable to carry out its normal functions. A particularly important enzyme that can act as a nucleophile is DNA polymerase, which is responsible for the synthesis of strands of DNA, especially as part of a DNA repair mechanism (see Section 14.2.2). The nucleophilic centre is a thiol grouping, and this may react with ethyl acrylate as shown. 

Acquired resistance to alkylating agents is a common event. Such resistance against the cytostatic activity can occur through at least three mechanisms. Increased thiol production can inactivate the agents. Also a decreased cell permeability to the drug can play a role. Increased capacity for DNA repair can mitigate cytotoxic activity. 

Possible biochemical mechanisms of resistance to alkylating agents include changes in ceU DNA repair capability, increases in cell thiol content (which in turn can serve as alternative and benign targets of alkylation), decreases in ceU permeability, and increased activity of glutathione transferases. Increased metaUothionein content has been associated with tumor cell resistance to cisplatin. 

Phenylbutazone. - This anti-inflammatory drug inhibits prostaglandin H synthase. Earlier spin-trapping studies established that PB is oxidised to a carbon-centred radical by the peroxidase activity of the enzyme.175 The radical has since been trapped with MNP upon incubation of the drug with HRP. The intensity of the signal from the adduct was reduced by GSH, suggesting chemical repair of the radical by the thiol. The PB/HRP system induced lipid peroxidation in microsomes, which was suppressed by GSH.176... 

The clinical usefulness of cis-Pt is often limited by the development of resistance. The development of resistance to cis-Pt has been explained by several factors, including reduced drug accumulation, increased DNA repair processes, and an increase in the amount of inactivation proteins. In most cis-Pt-resistant tumors probably a combination of such factors plays a role. In this section, only an increase in cellular thiols in relation to resistance will be discussed. Such inactivation processes have already been discussed (Section IV,B), but attention will be directed now to the mechanism of resistance. These mechanisms are probably also of importance for the inactivation of cis-Pt in nonre-sistant tumors, though to a lesser extent. 

Cells have two defense systems to cope with free-radical DNA damage that work on very different time scales the fast chemical repair by thiols that occurs at the stage of DNA free-radicals and the slow enzymatic repair that only sets in once the damage is fully set. The present book deals in some detail with the chemical repair. To discuss the even more important enzymatic repair would have exceeded the space allocated to this book, and enzymatic repair is only briefly touched on. 

The C-H BDE in peptides is even lower than that of the S-H BDE in thiols as a consequence of the exceptional stability of the radical products due to captoda-tive stabilization (Viehe et al. 1985 Armstrong et al. 1996). Yet, the observed rate constants for the reaction of CH3 and CH2OH with, e.g., alanine anhydride are markedly slower than with a thiol. This behavior has been discussed in terms of the charge and spin polarization in the transition state, as determined by AIM analysis, and in terms of orbital interaction theory (Reid et al. 2003). With respect to the repair of DNA radicals by neighboring proteins, it follows that the reaction must be slow although thermodynamically favorable. 

The (oxidizing) a-carboxyalkyl radicals do not react readily with thiols (Table 6.4). They are, however, rapidly reduced by thiolate ions [reaction (20)]. The reactions of thiols with DNA radicals play a very important role in the chemical repair of DNA radicals in cells (Chaps 12.10 and 12.11). The reversibility of the H-donation of thiols, that is, H-abstraction by thiyl radicals, is discussed in Chapter 7.4. 

The S-H bond is weak (alkylmercaptans BDE = 366 kj mol1, thiophenol BDE a 330 kj mol1 Armstrong 1999), and for this reason thiols can serve as H-donors [reaction (26)]. Thus thiols can play an important role in the repair of free-radi-cal-induced damage (for some early studies see Adams et al. 1967,1968, 1969). Some rate constants are compiled in Table 7.3. Compared to aqueous solutions, the rate of H-transfer by thiols is slower in organic solvents (Tronche et al. 1996). 

When the amino group is fully deprotonated, the rate of the H-transfer is 1.5 x 10s s4, but also around pH 7 the reaction is still fast, at the ms timescale (for a quantum mechanical study see Rauk et al. 2001). Upon the decay of the amnioal-kyl radicals formed in reaction (35) ammonia as formed in a yield that points to disproportionation as the major process (Zhao et al. 1997). The fact that the ami-noalkyl radical is the thermodynamically favored species does not mean that the repair of DNA radicals by GSH (Chap. 12.11) is not due to its action as a thiol. As with many other examples described in this book, the much faster kinetics that lead to a metastable intermediate (here the formation of the thiyl radical) rather than the thermodynamics as determined by the most stable species (here the aminoalkyl radical) determine the pathway the the reaction. In fact, the C-H BDE of the peptide linkage is lower than the S-H BDE and repair of DNA radicals by peptides, e.g., proteins would be thermodynamically favored over a repair by thiols but this reaction is retarded kinetically (Reid et al. 2003a,b). 

Prutz WA (1989) Chemical repair in irradiated DNA solutions containing thiols and/or disulphides. Further evidence for disulphide radical anions acting as electron donors. Int J Radiat Biol 56 21-33... 

With R = benzyl and in the absence of 02, the major product (73%) is the de-carbonylation product [reaction (209) possible formed to a large extent within the solvent cage], and the dimer of the allylic radical [reaction (207)] is formed only in small amounts. Addition of a thiol increases the yield of Thd [reaction (208)]. If an evaluation of the data reported for the reduction of the allylic OH-adduct to 1,3-cylohexadiene by a thiol (Pan et al. 1988), estimated at 104 dm3 mor1 s"1, is a good guide the rate constant for reaction (208) should be similar. This would revise an assumed rate constant of 106 dm3 mol-1 s-1 and the conclusions as to the repairability of allylic Thy in DNA radicals by cellular thiols (Anderson et al. 2000). 

O Neill P (1983) Pulse radiolytic study of the interaction of thiols and ascorbate with OH-adducts of dGMP and dG. Implications for DNA repair processes. Radiat Res 96 198-210 O Neill P (1984) Hydroxyl radical damage potential repair by sulphydryls, ascorbate and other antioxidants. Life Chem Rep Suppl Ser 2. In Rotilio G, Bannister JV (eds) Oxidative damage and related enzymes. Life Chem Rep 1 337-341... 

Table 11.4. Rate of repair of poly(U)-derived radicals by thiols at pH 7 and 4 (in parentheses). (Fahey et al. 1991) ...

With thiols, protection against free-radical attack and repair of DNA damage (Sect. 12.12) is not always easy to disentangle in in vitro experiments and even more so in cellular systems (e.g., Murray et al. 1990). This has to be kept in mind, when some aspects of thiol protections are discussed here. 

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