Redox Properties Of Phenols, Their Relationships To Singlet Oxygen Quenching And To Their Inhibitory Effects On Benzo(A)Pyrene-Induced Neoplasia

The inhibitory effects of synthetic phenolic compounds on benzo(a)pyrene-induced neoplasia of the mouse forestomach have been measured by Wattenberg et al⁶ The efficiency of this inhibition has been estimated for each phenol, using R, the ratio of the number of tumors per mouse in the protected group over the number of tumours per mouse in the control group. We have observed a linear correlation between the chemoprotection efficiency R and the logarithm of the rate of quenching of singlet oxygen. k. by this family of phenols, log k being itself correlated with the one-electron oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced hy benzo(a)py-rene. The correlations described provide a theoretical basis for scaling the inhibitors of mutagenicity induced by polycyclic aromatic compounds in terms of their oxidation potentials     

Redox Properties Of Phenols,Their Relationships To Singlet Oxygen Quenching And To Their Inhibitory Effects On Benzo(A)Pyrene-Induced Neoplasia

The inhibitory effects of synthetic phenolic compounds on benzo(a)pyrene-induced neoplasia of the mouse forestomach have been measured by Wattenberg et al⁶ The efficiency of this inhibition has been estimated for each phenol, using R, the ratio of the number of tumors per mouse in the protected group over the number of tumours per mouse in the control group. We have observed a linear correlation between the chemoprotection efficiency R and the logarithm of the rate of quenching of singlet oxygen. k. by this family of phenols, log k being itself correlated with the one-electron oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced hy benzo(a)py-rene. The correlations described provide a theoretical basis for scaling the inhibitors of mutagenicity induced by polycyclic aromatic compounds in terms of their oxidation potentials     

Relation of the antimutagenic activity of simple phenols to the number of hydroxyl groups

The influence of mono-phenol, di-resorcinol and tri-pyrogallol hydroxyl groups of simple unsubstituted phenols on the mutagenic potentials of benzo(a)pyrene was studied in vivo (micronuclear test on bone marrow polychromatic erythrocytes) and in vitro (test of direct point mutations at V79/HGPRT system induced by metabolic activation by mouse liver microsomal enzymes). The phenols decreased the mutagenic activity of benzo(a)pyrene in in vivo tests, with pyrogallol being the most active, it followed by resorcinol and phenol. The mixtures of benzo(a) pyrene + pyrogallol and benzo(a)pyrene + resorcinol were significantly less mutagenic in in vitro tests than benzo(a)pyrene and benzo(a)pyrene + phenol.     

UDP-glucuronosyl transferase and the conjugation of benzo(a)pyrene metabolites to DNA.

Addition of UDP-glucuronic acid to microsomal incubations containing benzo(a)pyrene caused a dose-dependent conjugation of principally quinone and phenol metabolites. Total benzo(a)pyrene oxidation was also stimulated with a maximum increase at 2 nM UDPGA. In the presence of calf thymus DNA, UDPGA caused a 2.7-fold increase in benzo(a)pyrene diol-oxide modification of DNA, as analyzed by Sephadex LH-20 chromatography. Maximum DNA modification by diol-oxides occurred at a UDPGA concentration which gave the highest level of free benzo(a)pyrene 7,8-dihydrodiol; likewise, the amount of DNA adduct derived from benzo(a)pyrene phenols declined in parallel with levels of free phenol metabolites. The UDPGA-induced increase in benzo(a)pyrene oxidation and concomitant increase in diol-oxide modification of DNA is consistent with removal of product inhibition by glucuronide conjugation of an inhibitory benzo(a)pyrene metabolite.     

Inhibition of AP-1 and neoplastic transformation by fresh apple peel extract

Consumption of fruits and vegetables has been associated with a low incidence of cancers and other chronic diseases. Previous studies suggested that fresh apples inhibit tumor cell proliferation. Here we report that oral administration of apple peel extracts decreased the number of nonmalignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. ESR analysis indicated that apple extract strongly scavenged hydroxyl (OH) and superoxide (O(2)(-)) radicals. Mechanistic studies showed that pretreatment with apple peel extract inhibited AP-1 transactivation induced by ultraviolet B irradiation or TPA in JB6 cells and AP-1-luciferase reporter transgenic mice. This inhibitory effect appears to be mediated by the inhibition of ERKs and JNK activity. The results provide the first evidence that an extract from fresh apple peel extract may inhibit tumor promoter-induced carcinogenesis and associated cell signaling, and suggest that the chemopreventive effects of fresh apple may be through its antioxidant properties by blocking reactive oxygen species-mediated AP-1-MAPK activation.     

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5‐S‐cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time‐resolved near‐infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450‐nm, quantum yield of singlet oxygen was very low (~10^?4) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.     

Reactions between Singlet Oxygen and the Constituents of Nucleic Acids: Importance of Reactions in Photodynamic Processes

Bases, nucleosides, nucleotides, and polynucleotides were exposed to chemically generated singlet oxygen to determine whether the species oxidized paralleled those oxidized in photodynamic reactions. In neutral or basic aqueous solution guanine, guanosine, deoxyguanosine, guanylic acid, deoxyguanylic acid, thymine, and uracil reacted with singlet oxygen. Since these compounds are oxidized in photodynamic processes, this study provides further evidence that singlet oxygen is the active intermediate in the photodynamic oxidation of nucleic acid constituents. Dienophilic attack by singlet oxygen is considered to be a plausible mechanism in these reactions.     

DNA strand scissions by hydroxamic acids in the presence of Cu(II) ion under aerobic conditions.

Carbazolyloxyacetohydroxamic acid (1), 9,9'-decamethylene-bis-carbazolyloxyacetohydroxamic acid (2), benzohydroxamic acid (3), and acetohydroxamic acid (4) without reducing agent under aerobic conditions induced DNA strand scissions with increasing activities in the order of 4 > 3 > 2 > 1. The inhibition experiments indicated that hydrogen peroxide and superoxide participated in the reactions, but hydroxyl radical or singlet oxygen did not.     

Benzo[a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2'-deoxyguanosine via reactive oxygen species

We previously reported that benzo[a]pyrene (BaP) and UVA radiation synergistically induced oxidative DNA damage via 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in vitro. The present study shows that microsomal BaP metabolites and UVA radiation potently enhance 8-OHdG formation in calf thymus DNA about 3-fold over the parent compound BaP. Utilization of various reactive oxygen species scavengers revealed that singlet oxygen and superoxide radical anion were involved in the 8-OHdG formation induced by microsomal BaP metabolites and UVA. Two specific BaP metabolites, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (+/-) (anti) (BPDE) and BaP-7,8-dione, were further tested for synergism with UVA. BaP-7,8-dione showed an effect on 8-OHdG formation induced by UVA radiation that was similar to that of the parent BaP, whereas BPDE exhibited significantly higher induction of 8-OHdG than BaP. At as low as 0.5 microM, BPDE plus UVA radiation substantially increased 8-OHdG levels about 25-fold over the parent BaP. BPDE increased the formation of 8-OHdG levels in both BPDE concentration- and UVA dose-dependent manners. Additionally, singlet oxygen was found to play a major role in 8-OHdG induction by BPDE and UVA. These results suggest that BaP metabolites such as BPDE synergize with UVA radiation to produce ROS, which in turn induce DNA damage.     

Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author's transl)

The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed.     

New Singlet Oxygen Source and Trapping Reagent for Peroxide Intermediates

Application of newly-developed water-soluble singlet oxygen sources in the oxidation of biologically important compounds and the electron-transfer process involving singlet oxygen has been reviewed. Particularly, oxidation products of tryptophan by chemically generated singlet oxygen were compared to those obtained in dye-sensitizd photooxygenation. The usefulness of trimethylsilyl cyanide as a trapping reagent for dipolar peroxide intermediates has been demonstrated in the photooxygenation of N-methylindoles, 2-(methoxymethylene)adamantane and adamantylideneadamantane in aprotic solvents. Based on these trapping reactions mechanism of singlet oxygen reaction of electron-rich enol ethers and enamines is discussed in light of theoretical calculation.     

New Singlet Oxygen Source and Trapping Reagent for Peroxide Intermediates

Application of newly-developed water-soluble singlet oxygen sources in the oxidation of biologically important compounds and the electron-transfer process involving singlet oxygen has been reviewed. Particularly, oxidation products of tryptophan by chemically generated singlet oxygen were compared to those obtained in dye-sensitizd photooxygenation. The usefulness of trimethylsilyl cyanide as a trapping reagent for dipolar peroxide intermediates has been demonstrated in the photooxygenation of N-methylindoles, 2-(methoxymethylene)adamantane and adamantylideneadamantane in aprotic solvents. Based on these trapping reactions mechanism of singlet oxygen reaction of electron-rich enol ethers and enamines is discussed in light of theoretical calculation.     

Initiation and inhibition of free-radical processes in biochemical peroxidase systems: a review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase- and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Coupled peroxidase-catalyzed oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (Ki, micromol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (alpha, M(-1)), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).     

In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.     

Roles of reactive oxygen species in UVA‐induced oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid‐melanin as studied by differential spectrophotometric method

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA‐induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA‐induced degradation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA‐induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole‐5,6‐quinone‐2‐carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole‐2,3,5‐tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA‐melanin was confirmed by direct measurements of singlet oxygen phosphorescence.     

Carotenoids as cellular antioxidants.

Consumption of carotenoids is associated with an enhanced immune response and protection against neoplasia and atherosclerosis. Because these effects have been achieved using carotenoids with no pro-vitamin A activity, they are assumed to be due to the antioxidant properties of carotenoids. Carotenoids protect against photosensitized oxidation by quenching singlet oxygen. In addition, beta-carotene reacts chemically with peroxyl radicals to produce epoxide and apocarotenal products. To investigate the potential significance of these reactions to biological systems, we have used soybean lipoxygenase to generate peroxyl radical enzymatically. beta-Carotene inhibits the oxidation of linoleic acid by soybean lipoxygenase as well as the formation of the hydroperoxide product. In addition, the absorption of beta-carotene is diminished (bleached) by soybean lipoxygenase. The potential significance of these antioxidant reactions of carotenoids to biological function is discussed.     

Oxidation of benzo(a)pyrene and 7,8-dihydro-7,8-dihydroxy benzo(a)pyrene by horseradish peroxidase-H2O2 intermediate: fluorometric study

The capacity of oxidation of benzo(a)pyrene (BP) and its analog to be oxidized by peroxidases in several tissues has been studied. The kinetics of the horseradish peroxidase (HRP) oxidation of BP and 7,8-dihydro-7,8-dihydroxy benzo(a)pyrene (BP-7,8-diol) were examined. Effective ratios of H2O2 and HRP for catalytic oxidation were 13.74 for BP and 4.58 for BP-7,8-diol. The maximum ratio was approximately 90 for both hydrogen donors (BP and BP-7,8-diol) to the ES complex. The maximum ratio of oxidized BP and BP-7,8-diol to HRP was 5.7. Ks values for H2O2 were 1.68 and 6.35 microM for BP and BP-7,8-diol, respectively. The mean values of the rate constants, k5, for the oxidation of BP and BP-7,8-diol were 0.56 X 10(5) M-1 sec-1 and 4.1 X 10(5) M-1 sec-1, respectively, at low concentrations. At low concentrations a Hill plot of the oxidation of BP showed a negative value (nH = 0.5) and at high concentrations nH = 1.0. On the other hand, that of BP-7,8-diol showed positive cooperativeness (nH = 1.8). These oxidation reactions caused substrate (donor) inhibition at high concentrations. The inhibition constants, KA', were 9.8 and 5.65 microM for BP and BP-7,8-diol, respectively. The reactivity of the oxidation of BP-7,8-diol was five to six times larger than that of BP.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Photodynamic properties of meta-tetra(hydroxyphenyl)chlorin in human tumor cells.

The photodynamic properties of a second-generation photodynamic sensitizer, meta-tetra(hydroxyphenyl)chlorin (mTHPC) were studied by dye-sensitized photoinactivation (650 nm) of HT29 human adenocarcinoma cells in culture. The photocytotoxicity of mTHPC in vitro depended on the presence of molecular oxygen. A strong inhibition of the photocytotoxicity of mTHPC was observed upon addition of sodium azide, a known singlet oxygen quencher. Photocytotoxicity was not inhibited by scavengers of superoxide anion radical, hydrogen peroxide and hydroxyl radicals. We suggest that mTHPC photosensitizes cell killing predominantly by type II, singlet oxygen-mediated photodynamic reactions. Illumination of cells preloaded with mTHPC induced peroxidation of membrane lipids. Inhibition of photoperoxidation by alpha-tocopherol (0.1 mM) present during illumination did not result in any decrease in toxicity, suggesting that reactions of lipid peroxidation play only a minor role in the overall photocytotoxic effect of mTHPC.     

血清胆红素的光敏氧化

竹红菌乙素是一种芘醌类的光敏剂.实验结果表明在可见光的照射下.它能加速血清胆红素的光氧化,氧化速率提高5倍以上.比较在不同溶剂中各种活性氧淬灭剂对胆红素光氧化的抑制作用,指出血清胆红素光敏氧化反应包括自由基氧化反应(Ⅰ型反应)和单态氧氧化反应(Ⅱ型反应)等多重机制.