Oxidative Degradation Of DNA Research Articles

8-Hydroxy-2-deoxyguanosine, a product of oxidative DNA degradation, is increased in the amniotic fluid of preterm births.

8-Hydroxy-2-deoxyguanosine (8-OH-2dG) is a measurable biomarker of oxidative DNA damage. This study was designed to determine amniotic fluid 8-OH-2dG levels in healthy full-term pregnant women and preterm pregnant women. To reveal the effect of reactive oxygen species on 8-OH-2dG levels, amniotic fluid total oxidant capacity (TOS), total antioxidant capacity (TAC) and oxidative stress index (OSI) were also measured. A total of 60 patients, 35 patients with full-term pregnancy and 25 patients with preterm pregnancy, participated in the study. Labor occurring before 37 weeks of gestation was considered as spontaneous preterm birth. Amniotic fluid samples were collected from full-term patients during cesarean section or normal vaginal delivery. 8-OH-2dG concentrations in amniotic fluid samples were measured quantitatively by Enzyme-Linked Immunosorbent Assay (ELISA). Amniotic fluid total antioxidant capacity (TAC) and total oxidant capacity (TOC) was determined in amniotic samples. The amniotic fluid 8-OH-2dG levels of the preterm group were significantly higher than the full-term group (60.8±7.02 ng/mL vs. 33.6±4.11 ng/mL, p<0.01). Similarly, TOC levels of the preterm group were significantly higher than the full-term group (89.7±4.80 µmol/L vs. 54.3±6.60 µmol, p<0.02). TAC was significantly higher in the full-term group compared to the preterm group (1.87±0.10 mmol/L vs. 0.97±0.44 mmol/L, p<0.01). The OSI values of the preterm group were significantly higher than the full-term group. A negative and significant correlation was found between gestational age and amniotic fluid 8-OH-2dG levels in the full-term pregnancy group (r=-0.78, p<0.01). A negative and significant correlation was observed between TAC and amniotic fluid 8-OH-2dG levels in the full-term group (r=-0.60, p<0.02). A positive and significant correlation was also detected between TOC, OSI and amniotic fluid 8-OH-2dG levels in the full-term group. There was a negative but insignificant correlation between fetal weight and amniotic fluid 8-OH-2dG levels. The correlation analysis results of the preterm pregnancy group were similar to the full-term group. Increased reactive oxygen derivatives in preterm birth increase amniotic fluid levels of DNA degradation product 8-OH2dG and may lead to premature rupture of fetal membranes. This is the first clinical study investigating 8-OH-2dG levels in amniotic fluid of preterm birth.

Impact of N-heteroaromatic N-termini in Cu(II) ATCUN metallopeptides on their biorelevant redox activity.

Cu(II) complexes with ATCUN peptide ligands have been investigated for their ROS (reactive oxygen species) generation and oxidative DNA degradation abilities. The biological activity of most ATCUN complexes such as Cu-GGH (Gly-Gly-His) is, however, low. Tuning the redox chemistry by incorporation of N-heteroaromatics reinstates ROS production which leads to efficient DNA cleavage.

Structural and Functional Studies of S-(2-Carboxyethyl)-L-Cysteine and S-(2-Carboxyethyl)-l-Cysteine Sulfoxide.

Insecticidal non-proteinogenic amino acid S-(2-carboxyethyl)-L-cysteine (β-CEC) and its assumed metabolite, S-(2-carboxyethyl)-l-cysteine sulfoxide (β-CECO), are present abundantly in a number of plants of the legume family. In humans, these amino acids may occur as a result of exposure to environmental acrylonitrile or acrylamide, and due to consumption of the legumes. The β-CEC molecule is a homolog of S-carboxymethyl-l-cysteine (carbocisteine, CMC), a clinically employed antioxidant and mucolytic drug. We report here detailed structural data for β-CEC and β-CECO, as well as results of in vitro studies evaluating cytotoxicity and the protective potential of the amino acids in renal tubular epithelial cells (RTECs) equipped with reporters for activity of seven stress-responsive transcription factors. In RTECs, β-CEC and the sulfoxide were not acutely cytotoxic, but activated the antioxidant Nrf2 pathway. β-CEC, but not the sulfoxide, induced the amino acid stress signaling, which could be moderated by cysteine, methionine, histidine, and tryptophan. β-CEC enhanced the cytotoxic effects of arsenic, cadmium, lead, and mercury, but inhibited the cytotoxic stress induced by cisplatin, oxaliplatin, and CuO nanoparticles and acted as an antioxidant in a copper-dependent oxidative DNA degradation assay. In these experiments, the structure and activities of β-CEC closely resembled those of CMC. Our data suggest that β-CEC may act as a mild activator of the cytoprotective pathways and as a protector from platinum drugs and environmental copper cytotoxicity.

Ruthenium(II)–arene complexes of diimines: Effect of diimine intercalation and hydrophobicity on DNA and protein binding and cytotoxicity

A series of half‐sandwich Ru(II)–arene complexes [Ru(η6‐benzene)(diimine)Cl](PF6) (1–4), where diimine is 1,10‐phenanthroline (1), 5,6‐dimethyl‐1,10‐phenanthroline (2), dipyrido[3,2‐a:2′,3′‐c]phenazine (3) or 11,12‐dimethyldipyrido[3,2‐a:2′,3′‐c]phenazine (4), have been isolated and characterized using analytical and spectral methods. Complex 2 possesses a familiar pseudo‐octahedral ‘piano‐stool’ structure. The intrinsic DNA binding affinity of the complexes depends upon the diimine ligand: 3 (dppz) &gt; 4 (11,12‐dmdppz) &gt; 2 (5,6‐dmp) &gt; 1 (phen). The π‐stacking interaction of extended planar ring of coordinated dppz (3) in between the DNA base pairs is more intimate than that of phen (1), and the incorporation of methyl groups on the dppz ring (4) discourages the stacking interaction leading to a lower DNA binding affinity for 4 than 3. Docking studies show that all the complexes bind in the major groove of DNA. Interestingly, 3 shows an ability to convert supercoiled DNA into nicked circular DNA even at 20 μM concentration beyond which complete oxidative DNA degradation is observed. The protein binding affinity of the complexes decreases in the order 4 &gt; 3 &gt; 2 &gt; 1, and the higher protein binding affinity of 4 illustrates the strong involvement of methyl groups on dppz ring in hydrophobic interaction with protein. Also, 4 cleaves protein more efficiently than the other complexes in the presence of H2O2. It is notable that 2, 3 and 4 display cytotoxicity against human cervical cancer cell lines (SiHa) with potency higher than the currently used drug cisplatin. Acridine orange/ethidium bromide staining studies reveal that 3 induces apoptosis in cancer cells much more efficiently than 4.

Mobilization of Nuclear Copper by Green Tea Polyphenol Epicatechin-3-Gallate and Subsequent Prooxidant Breakage of Cellular DNA: Implications for Cancer Chemotherapy.

Epidemiological as well as experimental evidence exists in support of chemopreventive and anticancer properties of green tea and its constituents. The gallocatechin, epicatechin-3-gallate is a major polyphenol present in green tea, shown responsible for these effects. Plant-derived polyphenolic compounds are established natural antioxidants which are capable of catalyzing oxidative DNA degradation of cellular DNA, alone as well as in the presence of transition metal ions, such as copper. Here we present evidence to support that, similar to various other polyphenoic compounds, epicatechin-3-gallate also causes oxidative degradation of cellular DNA. Single cell alkaline gel electrophoresis (Comet assay) was used to assess DNA breakage in lymphocytes that were exposed to various concentrations of epicatechin-3-gallate. Inhibition of DNA breakage in the presence of scavengers of reactive oxygen species (ROS) suggested involvement of ROS generation. Addition of neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation, dose-dependently, in intact lymphocytes. In contrast, bathocuproine, which does not permeate cell membrane, was observed to be ineffective. We further show that epicatechin-3-gallate degrades DNA in cell nuclei, which can also be inhibited by neocuproine, suggesting mobilization of nuclear copper in this reaction as well. Our results are indicative of ROS generation, possibly through mobilization of endogenous copper ions, and support our long-standing hypothesis of a prooxidant activity of plant-derived polyphenols as a mechanism for their documented anticancer properties.

The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions.

We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

Nutritive and Dietetic Value of Genetically Modified Tomatoes Expressing Thaumatin Gene

Genetically-modified (GM) tomatoes, carrying thaumatin gene encoding sweet-tasting protein may be a component of diet with high sensory values, constituting a valuable source of nutrients and substances with a health-promoting role. Good utilization and a lack of the effect on animal growth, value of hematological parameters, concentration of immunoglobulins and most of chemical blood parameters of laboratory rats were demonstrated in the nutritional studies on fruits of tomato GM plants. The biological response of the rats receiving GMO or its isogenic equivalent in the diet was recognized as similar. However, the unfavourable effect of the diets containing addition of tomatoes with the recombined thaumatin on the degree of oxidative degradation of DNA of rats liver was recorded. At the same time, the discussed dietary component had no effect on values of the remaining parameters of the oxidative status of tissue of the above mentioned organ and its histological image.

Mapping structurally defined guanine oxidation products along DNA duplexes: influence of local sequence context and endogenous cytosine methylation.

DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along p53 gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2H-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated MeCG dinucleotides and at 5′ Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of MeCG sequences may be caused by a lowered ionization potential of guanine bases paired with MeC and the preferential intercalation of riboflavin photosensitizer adjacent to MeC:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known p53 lung cancer mutational “hotspots” at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer.

Discovery and Mutagenicity of a Guanidinoformimine Lesion as a new Intermediate of the Oxidative Deoxyguanosine Degradation Pathway

Oxidative degradation of DNA is a major mutagenic process. Reactive oxygen species (ROS) produced in the course of oxidative phosphorylation or by exogenous factors are known to attack preferentially deoxyguanosine. The latter decomposes to give mutagenic lesions, which under physiological conditions are efficiently repaired by specialized maintenance systems in the cell. Although many intermediates of the degradation pathway are today well-known, we report in this study the discovery of a new intermediate with an interesting guanidinoformimine structure. The structure elucidation of the new lesion was possible by using HPLC-MS techniques and organic synthesis. Finally we report the mutagenic potential of the new lesion in comparison to the known lesions imidazolone and oxazolone using primer extension and pyrosequencing experiments.

Prevention of Oxidative DNA Degradation by Copper-Binding Peptides

Free divalent ions of copper (Cu) are capable of generating radical species such as hydroxyl radicals in the presence of hydrogen peroxide or ascorbic acid through Harbor-Weiss-like reactions under physiological conditions. It has been reported that radical-mediated damage to DNA molecules in animal cells leads to programmed cell death. Hence it is important to seek for methods to prevent Cu-mediated DNA damage. In this study we identified on effect of Cu binding of short peptides (chiefly Gly-Gly-His tripeptide) in the prevention of DNA degradation caused by Cu-mediated reactions in the presence of hydrogen peroxide and of ascorbic acid.

Charge Separation and Photosensitized Damage in DNA Mediated by Naphthalimide, Naphthaldiimide, and Anthraquinone

Charge-separation and charge-recombination dynamics and oxidative DNA degradation were investigated for DNA modified with a photosensitizer (Sens) naphthalimide (NI), naphthaldiimide (ND), or anthraquinone (AQ). In all three Sens-modified DNA systems, the formation of long-lived charge-separated states was observed in which the lifetime increased with increasing numbers of A-T base pairs between Sens and the neighboring G-C base pair. The lifetime of the charge-separated state correlated well with the DNA damage yield, indicating that the charge-separated state provides time for the irreversible DNA oxidative damage to occur. The quantum yield of DNA damage was the lowest for ND-modified DNA due to the slow reaction of ND radical anion with molecular oxygen; the process needed to preclude charge recombination. The AQ-modified DNA resulted in the highest charge separation and subsequent DNA damage yield, which would be partly explained by the formation of the spin-forbidden triplet radical ion pairs during charge separation.

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 μM) for 1 h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties.

Resveratrol–Cu(II) induced DNA breakage in human peripheral lymphocytes: Implications for anticancer properties

Resveratrol (3,4′,5-trihydroxy stilbene), a plant derived polyphenol found in mulberries, grapes and red wine is considered to possess chemopreventive properties against cancer. It is recognized as a naturally occurring antioxidant but also catalyzes oxidative DNA degradation in vitro in the presence of transition metal ions such as copper. Using a cellular system of lymphocytes isolated from human peripheral blood and Comet assay, we have confirmed that resveratrol–Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. Also, trans-stilbene, which does not have any hydroxyl groups, is inactive in the lymphocyte system. Pre-incubation of lymphocytes with resveratrol indicates that it is capable of either traversing the cell membrane or binding to it. Our results are in partial support of our hypothesis that anticancer properties of various plant derived polyphenols may involve mobilization of endogenous copper and the consequent prooxidant action.

Prooxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: implications for anticancer properties

It is believed that anticancer and apoptosis inducing properties of green tea are mediated by it's polyphenolic constituents particularly catechins. A number of reports have shown that green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) is among the most effective chemopreventive and apoptosis-inducing agents present in the beverage. Plant polyphenols are naturally occurring antioxidants but they also exhibit prooxidant properties. Over the last several years we have shown that various classes of plant polyphenols including flavonoids, curcuminoids and tannins are capable of catalyzing oxidative DNA cleavage particularly in the presence of transition metal ions such as copper and iron. With a view to understand the chemical basis of various pharmacological properties of green tea, in this paper we have compared the prooxidant properties of green tea polyphenols––EGCG and EC ((−)-epicatechin). The rate of oxidative DNA degradation as well as hydroxyl radical and superoxide anion formation was found to be greater in the case of EGCG as compared with EC. It was also shown that copper mediated oxidation of EC and EGCG possibly leads to the formation of polymerized polyphenols. Further, it was indicated that copper oxidized catechins were more efficient prooxidants as compared with their unoxidized forms. These results correlate with the observation by others that EGCG is the most effective apoptosis inducing polyphenol present in green tea. They are also in support of our hypothesis that prooxidant action of plant polyphenols may be an important mechanism of their anticancer properties. A model for binding of Cu(II) to EC has been presented where the formation of quinone and a quinone methide has been proposed.

Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage.

Epidemiological evidence suggests that certain paternal exposures to metals may increase the risk of cancer in the progeny. This effect may be associated with promutagenic damage to the sperm DNA. The latter is packed with protamines which might sequester carcinogenic metals and moderate the damage. Human protamine P2 has an amino acid motif at its N-terminus that can serve as a heavy metal trap, especially for Ni(II) and Cu(II). We have synthesized a pentadecapeptide modeling this motif, Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP21-15) and described its complexes with Ni(II) and Cu(II), including their capacity to mediate oxidative DNA degradation [Bal et al. (1997) Chem. Res. Toxicol., 10, 906-914 and 915-921]. In the present study, effects of HP21-15 on Ni(II)- and Cu(II)-mediated DNA oxidation by H2O2 at pH 7.4 were investigated in more detail using the circular plasmid pUC19 DNA as a target, and the single/double-strand breaks and production of oxidized DNA bases, as end points. Ni(II) alone was found to promote oxidative DNA strand scission (mostly single strand breaks) and base damage, while Cu(II) alone produced the same effects, but to a much greater extent. Both metals were relatively more damaging to the pyrimidine bases than to purine bases. HP21-15 tended to increase the Ni(II)/H2O2-induced DNA breakage. In sharp contrast, the destruction of DNA strands by Cu(II)/H2O2 was almost completely prevented by HP21-15. The effect of HP21-15 on the oxidative DNA base damage varied from a limited enhancement (5-hydroxyhydantoin and thymine glycol) to slight suppression (5-hydroxycytosine, 5-hydroxyuracil, 8-oxoguanine, 8-oxoadenine, 2-hydroxyadenine, fapyguanine and fapyadenine) toward Ni(II)/H2O2. HP21-15 strongly suppressed the oxidative activity of Cu(II)/H2O2 in regard to all bases in DNA. Consistently with the above, the electron spin resonance/spin trap measurements revealed greater and more persistent generation of OH* and O2-*-like oxidants from H2O2 by the Ni(II)-HP21-15 complex than by the Cu(II)-HP21-15 complex (no O2-* was detected). Both complexes were also found to bind to DNA more strongly than HP21-15 alone. The results indicate that protamine P2 is capable of binding Ni(II) and Cu(II) and, in this way, attenuating the mediation of oxidative DNA damage by Cu(II), but not Ni(II). The effects found may be mechanistically involved in the reproductive toxicity and carcinogenicity of metals.

Extracts of Bolivian plants,Copaifera reticulataandHeisteria pallidainhibitin vitrofree radical-mediated DNA damage

The presence of different extracts of antiinflammatory plants Copaifera reticulata and Heisteria pallida in a reaction medium containing calf thymus DNA in a free radical generating system protected DNA against oxidative damage in terms of deoxyribose oxidation. The highest antioxidant activity was obtained using the methanol extract of C. reticulata (IC50=3 μg/mL), followed by the aqueous extracts of H. pallida (IC50=257 μg/mL) and C. reticulata (IC50=380 μg/mL). Both dichloromethane extracts and the methanol extract of H. pallida showed a decreased antioxidant activity at higher concentrations. These results suggest that these extracts are capable of suppressing the in vitro oxidative degradation of DNA. © 1997 John Wiley & Sons, Ltd.

Oxidative DNA damage induced by dioxetanes, photosensitizing ketones, and photo-fenton reagents.

In the last decade, the importance of oxidative DNA damage in mutagenesis, carcinogenesis, aging, and various diseases has prompted intensive investigations of chemical, biochemical, and biological aspects of DNA oxidation caused by reactive species, which are involved in oxidative stress (Sies 1991). Oxidative degradation of DNA causes mutations predominantly at GC base pairs to yield single base substitutions and G to T transversions (Piette 1991). Consequently, the DNA transformation efficiency is diminished and replication is inhibited.

Inhibition of lipid peroxidation and iron (II)-dependent DNA damage by extracts of Pothomorphe peltata (L). Miq.

Leaves of Pothomorphe peltata (L.) Miq. (Piperaceae) are used locally as anti-inflammatory, antipyretic, hepatoprotective and diuretic infusions and to treat external ulcers and local infections in several parts of the Peruvian, Bolivian and Brazilian Amazon region. The antioxidant activity of different extracts of P. peltata was studied using the hydroperoxide-initiated chemiluminescence assay in liver homogenates, and the methanolic extract was found to have the highest antioxidant activity, with an IC50 = 4 micrograms/ml. Aqueous and dichloromethane extracts did not show antioxidant activity. The extracts were further evaluated using the thiobarbituric acid-reactive substances (TBARS) assay. Finally, an assay of DNA sugar damage induced by Fe (II) salt was used to determine the capacity of the extracts to suppress the oxidative degradation of DNA. All the extracts showed antioxidant activity in the latter two bioassays. The methanolic extract showed the highest activity in reducing oxidative damage to DNA, with an IC50 = 5 micrograms/ml. Since this extract was highly effective in reducing chemiluminescence and DNA damage, and because the latter activity could be due to the presence of compounds that bind to DNA, DNA-binding activity was studied using the DNA-methyl green (DNA-MG) bioassay. A 30% decrease in the initial absorbance of DNA-MG complex was observed in the methanolic extract at 1000 micrograms/ml, suggesting the presence of compounds that bind to genetic material. No DNA-binding activity was observed in the aqueous or dichloromethane extracts.

Sequence-specific hydrolysis of yeast tRNA(Phe) mediated by metal-free bleomycin.

Bleomycin A2 (BLM) was found to mediate sequence specific hydrolysis of tRNA(Phe) in the absence of added metal ions. BLM A2 promoted phosphodiester bond hydrolysis 3' to the pyrimidine residue at all resolved Py-Pu sites not involving modified bases, as demonstrated by high-resolution electrophoretic analysis of 5'- and 3'-32P-end-labeled substrates. The reaction proceeded with surprising facility, approaching efficiency that of oxidative strand scission mediated by the FeII.BLM A2 complex. By the use of a number of BLM congeners, as well as a study of the time, temperature, and salt dependence of the hydrolysis, it was shown that in many respects the hydrolytic reaction parallels the oxidative degradation of RNA and DNA mediated by metallobleomycins. Thus, in contrast to the well-characterized oxidative degradation of DNA and RNA by bleomycin studied for two decades, the present report documents the ability of certain metal-free bleomycins to mediate RNA hydrolysis.

Phenoxyl Radicals of Etoposide (VP-16) Can Directly Oxidize Intracellular Thiols: Protective Versus Damaging Effects of Phenolic Antioxidants

Phenolic compounds can act as radical scavengers due to their ability to donate a mobile hydrogen to peroxyl radicals producing a phenoxyl radical if the phenoxyl radical formed in the radical scavenging reaction efficiently interacts with vitally important biomolecules, then this interaction may result in cytotoxic effects rather than in antioxidant protection. In the present work we have chosen two model compounds--a phenolic antitumor drug, VP-16, known to be highly cytotoxic, and a homolog of vitamin E, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC)--as typical representatives of phenoxyl radicals to study interactions of their phenoxyl radicals with intracellular thiols. Using a water-soluble source of peroxyl radicals, the azo-initiator 2,2'-azobis(2-aminodinopropane) (AAPH), we found that both PMC and VP-16 are very efficient scavengers of peroxyl radicals as evidenced by their ability to inhibit AAPH-induced chemiluminescence of luminol and oxidation of PnA incorporated into DOPC liposomes. Both PMC and VP-16 were also able to protect against AAPH-induced oxidative degradation of DNA in nuclei from human leukemic K562 cells. In contrast, there was a dramatic difference in the ability of VP-16 and PMC to protect GSH against AAPH-induced oxidation: while PMC inhibited AAPH-induced oxidation of GSH in a concentration-dependent manner, VP-16 did not protect GSH against oxidation. We hypothesized that this was due to different reactivities of the phenoxyl radicals formed by AAPH-derived peroxyl radicals from VP-16 and PMC toward GSH. To substantiate this hypothesis, we compared interactions of the phenoxyl radicals generated from VP-16 and PMC with intracellular thiols in K562 cell homogenates. While the PMC phenoxyl radicals were only slightly affected by thiols, the VP-16 phenoxyl radicals were reduced by thiols. This is evidenced by (i) a significant inhibition of the tyrosinase-induced VP-16 consumption upon addition of K562 cell homogenates, (ii) a depletion of endogenous thiols in K562 cell homogenates induced by VP-16+tyrosinase, (iii) a transient disappearance of the VP-16 phenoxyl radical signal from the ESR spectra and its reappearance after depletion of endogenous thiols, and (iv) elimination of the lag period for the appearance of the VP-16 phenoxyl radical ESR signal subsequent to depletion of thiols by mersalyl acid. To evaluate the contribution of GSH and protein thiols to reduction of the VP-GSH-peroxidase + cumeme hydroperoxide to specifically deplete endogenous GSH.(ABSTRACT TRUNCATED AT 400 WORDS)