Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention

Epidemiological studies have suggested that the use of aspirin is associated with a decreased incidence of human malignancies, particularly colorectal cancer. Since reactive oxygen species (ROS) are critically involved in multistage carcinogenesis, this study was undertaken to examine the ability of aspirin to inhibit ROS-mediated DNA damage. Hydrogen peroxide (H2O2)+Cu(II) and hydroquinone (HQ) + Cu(II) were used to cause oxidative DNA strand breaks in phiX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.5-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a marked inhibition of oxidative DNA damage induced by either H2O2/Cu(II) or HQ/Cu(II). The inhibition of oxidative DNA damage by aspirin was exhibited in a concentration-dependent manner. Moreover, aspirin was found to be much more potent than the hydroxyl radical scavengers, mannitol and dimethyl sulfoxide, in protecting against the H2O2/Cu(II)-mediated DNA strand breaks. Since the reduction of Cu(II) to Cu(I) is crucially involved in both H2O2/Cu(II)- and HQ/Cu(II)-mediated formation of hydroxyl radical or its equivalent, and the subsequent oxidative DNA damage, we examined whether aspirin could inhibit this Cu(II)/Cu(I) redox cycle. It was observed that aspirin at concentrations that showed the inhibitory effect on oxidative DNA damage did not alter the Cu(II)/Cu(I) redox cycle in either H2O2/Cu(II) or HQ/Cu(II) system. In addition, aspirin was not found to significantly scavenge H2O2. This study demonstrates for the first time that aspirin potently inhibits both H2O2/Cu(II)- and HQ/Cu(II)-mediated oxidative DNA strand breaks most likely through scavenging the hydroxyl radical or its equivalent derived from these two systems. The potent inhibition of oxidative DNA damage by aspirin may thus partially contribute to its anticancer activities observed in humans.     

Copper redox-dependent activation of 2-tert-butyl(1,4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes

The biotransformation of butylated hydroxyanisole (BHA), a possible carcinogenic food antioxidant, includes o-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) which can subsequently be oxidized to 2-tert-butyl(1,4)paraquinone (TBQ). In this study, we have examined the capacity of Cu, a nuclei- and DNA-associated transition metal, to mediate the oxidation of TBHQ. In phosphate buffered saline (PBS), autooxidation of TBHQ to TBQ was not detectable, while Cu(II) at micromolar concentrations strongly catalyzed the oxidation of TBHQ to TBQ. Oxidation of TBHQ by Cu(II) was accompanied by the utilization of O(2) and the concomitant generation of H(2)O(2). Using electron spin resonance spectroscopy, it was observed that Cu(II) mediated the one electron oxidation of TBHQ to a semiquinone anion radical. The formation of a semiquinone anion radical, the utilization of O(2) and the generation of H(2)O(2) and TBQ could be completely blocked by bathocuproinedisulfonic acid (BCS) and reduced glutathione (GSH), two Cu(I)-chelators. 4-Pyridyl-1-oxide-N-tert-butylnitrone (POBN)-spin trapping experiments showed that the reaction of TBHQ with Cu(II) resulted in the generation of POBN-CH(3) and POBN-CH(OH)CH(3) adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting the formation of hydroxyl radical or a similar reactive intermediate. The formation of POBN-CH(3) adduct from the TBHQ/Cu(II)+DMSO could be completely inhibited by catalase, GSH or BCS, indicating that the hydroxyl radical or its equivalent is generated from the interaction of H(2)O(2) with Cu(I). Incubation of supercoiled phiX-174 plasmid DNA with the TBHQ/Cu(II) resulted in extensive DNA strand breaks, which could be prevented by catalase or BCS. Incubation of rat hepatocytes with TBHQ in PBS led to increased formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in nuclear DNA. The TBHQ-induced formation of 8-OHdG was markedly reduced in the presence of cell permeable Cu(I)-specific chelator, bathocuproine or neocuproine, suggesting that a Cu(II)/Cu(I) redox mechanism may also be involved in the induction of oxidative DNA damage by TBHQ in hepatocytes. Taken together, the above results conclusively demonstrate that the activation of TBHQ by Cu(II) results in the formation of TBQ, semiquinone anion radical and reactive oxygen species (ROS), and that the ROS formed may participate in oxidative DNA damage in both isolated DNA and intact cells. These reactions may contribute to the carcinogenicity as well as other biochemical activities observed with BHA in animals. To our knowledge this study provides the first evidence that endogenous cellular Cu may be capable of bioactivating TBHQ, leading to oxidative DNA damage in cultured cells.     

Thiols can either enhance or suppress DNA damage induction by catecholestrogens

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.     

Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide

Oxidative DNA damage has been implicated in diverse biological processes including mutagenesis, carcinogenesis, aging, radiation effects, and chemotherapy. We examined the in vitro effect of low concentrations of Cu(II) or H2O2 alone and in combination on supercoiled plasmid DNA. As much as 10(-2) M Cu(II) or 10(-2) M H2O2 alone did not break the DNA. However, a mixture of 10(-6) M Cu(II) plus 10(-5) M H2O2 produced strand breaks and inactivated transforming ability. Strand breakage was proportional to incubation time, temperature, and Cu(II) and H2O2 concentrations. Abasic sites were not detected. Strand breakage was inhibited by metal chelators, catalase, and by high levels of free radical scavengers implying that Cu(II), Cu(I), H2O2, and .OH were involved in the reaction. The extent of DNA strand breakage was not affected by superoxide dismutase indicating that superoxide was not a major contributor to the DNA damage. DNA sequence analysis demonstrated that hot piperidine-sensitive DNA lesions were produced preferentially at sites of 2 or more adjacent guanosine residues. This sequence specificity was observed with Cu(II) plus H2O2 but not with Cu(I) alone. Polyguanosine sequence specificity for DNA damage induction appears to be unique among simple chemical systems. This reaction may be important in mechanisms of oxidative damage in vivo.     

DNA and RNA damage by Cu(II)-amikacin complex.

The oxidation-promoting reactivity of copper(II) complex of aminoglycosidic antibiotic amikacin [Cu(II)-Ami] in the presence of hydrogen peroxide, was studied at pH 7.4, using 2'-deoxyguanosine (dG), pBR322 plasmid DNA and yeast tRNAPhe as target molecules. The mixtures of complex with H2O2 were found to be efficient oxidants, converting dG to its 8-oxo derivative, generating strand breaks in plasmid DNA and multiple cleavages in tRNAPhe. The complex underwent autooxidation as well, with amikacin hydroperoxides as likely major products. This reactivity pattern was found to be due to a combination of metal-bound and free hydroxyl radicals.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

In vitro oxidative activity of cupric complexes of kanamycin A in comparison to in vivo bactericidal efficacy

The interactions of copper(II) complexes of kanamycin A with oxidation-susceptible biomolecules: 2'-deoxyguanosine, plasmid DNA and yeast tRNA(Phe) were studied in both the presence and absence of hydrogen peroxide. The mixture of complex with H(2)O(2) was found to be an efficient oxidant, converting dG to its 8-oxo derivative, generating strand breaks in plasmid DNA and multiple cleavages in tRNA(Phe). Some of these reactions may play a role in toxic effects of aminoglycoside antibiotics. These complexes were screened for their antibacterial activity. The microbiological studies undertaken to compare the bactericidal action of kanamycin A alone and complexed with copper(II) ions in both neutral and oxidative environment revealed that the enhancement of bactericidal action by Cu(II) was not statistically significant.     

Copper-activated DNA photocleavage by a pyridine-linked bis-acridine intercalator

We report the synthesis of new photonuclease 4 consisting of two acridine rings joined by a pyridine-based copper binding linker. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of copper(II) (419 nm, 22 degrees C, pH 7.0). Viscometric data indicate that 4 binds to DNA by monofunctional intercalation, and equilibrium dialysis provides an estimated binding constant of 1.13 x 105 M-1 for its association with calf thymus DNA. In competition dialysis experiments, 4 exhibits preferential binding to GC-rich DNA sequences. When Cu(II) is added at a ligand to metal ratio of 1:1, electrospray ionization mass spectrometry demonstrates that compound 4 undergoes complex formation, while thermal melting studies show a 10 degrees C increase in the Tm of calf thymus DNA. Groove binding and intercalation are suggested by viscometric data. Finally, colorimetric and scavenger experiments indicate that the generation of Cu(I), H2O2, and superoxide contributes to the production of DNA frank strand breaks by the Cu(II) complex of 4. Whereas the strand breaks are distributed in a relatively uniform fashion over the four DNA bases, subsequent piperidine treatment of the photolysis reactions shows that alkaline labile lesions occur predominantly at guanine.     

DNA damage induced by catecholestrogens in the presence of copper (II): generation of reactive oxygen species and enhancement by NADH

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.     

DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: mechanism and structure-activity relationship

The prooxidant effect of resveratrol (3,5,4′-trihydroxy-trans-stibene) and its synthetic analogues (ArOH), that is, 3,4,4′-trihydroxy-trans-stibene (3,4,4′-THS), 3,4,5-trihydroxy-trans-stibene (3,4,5-THS), 3,4-dihydroxy-trans-stibene (3,4-DHS), 4,4′-dihydroxy-trans-stibene (4,4′-DHS), 2,4-dihydroxy-trans-stilbene (2,4-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-trans-stibene (3,5,4′-TMS), on supercoiled pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions has been studied. It was found that the compounds bearing ortho-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) or bearing 4-hydroxyl groups (2,4-DHS, 4,4′-DHS, and resveratrol) exhibit remarkably higher activity in the DNA damage than the ones bearing no such functionalities. Kinetic analysis by UV-visible spectra demonstrates that the formation of ArOH-Cu (II) complexes, the stabilization of oxidative intermediate derived from ArOH and Cu (II)/Cu (I) redox cycles, might be responsible for the DNA damage. This study also reveals a good correlation between antioxidant and prooxidant activity, as well as cytotoxicity against human leukemia (HL-60 and Jurkat) cell lines. The mechanisms and implications of these observations are discussed.     

Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase.

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.     

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

A mechanistic study on the nuclease activities of some hydroxystilbenes

The nuclease activities of a series of hydroxystilbeneoids have been studied so as to establish a structure-activity correlation and deduce the mechanistic pathway of the process. Although the test compounds could nick plasmid DNA, only three of these including resveratrol produced double strand breaks in DNA. Amongst these new stilbenes, compound 2e containing a partially methylated catechol and a C-4 hydroxy moieties was equally potent as resveratrol. The activities of the unprotected catechol-derivatives were less than those of the resorcinol-derivatives, which were, however, compensated by partial methylation of the former. The presence of Cu(2+) and O(2) and the participation of a Cu(+)-oxo intermediate were obligatory in the process which did not require addition of any external reducing agent. Overall, the differential nuclease activities of the compounds could be explained primarily with their superoxide anion generation abilities, and to a lesser extent with their DNA binding and Cu(2+) reducing capacities. The amount of superoxide anion produced by the compounds depended strongly on their Cu(+)-complexation abilities, which again, was decided by the pattern and nature of the oxygenated substituents in the aromatic rings.     

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his(+) reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H(2)O(2)/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H(2)O(2)/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H(2)O(2) formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.     

DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: Mechanism and structure-activity relationship

The prooxidant effect of resveratrol (3,5,4′-trihydroxy-trans-stibene) and its synthetic analogues (ArOH), that is, 3,4,4′-trihydroxy-trans-stibene (3,4,4′-THS), 3,4,5-trihydroxy-trans-stibene (3,4,5-THS), 3,4-dihydroxy-trans-stibene (3,4-DHS), 4,4′-dihydroxy-trans-stibene (4,4′-DHS), 2,4-dihydroxy-trans-stilbene (2,4-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-trans-stibene (3,5,4′-TMS), on supercoiled pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions has been studied. It was found that the compounds bearing ortho-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) or bearing 4-hydroxyl groups (2,4-DHS, 4,4′-DHS, and resveratrol) exhibit remarkably higher activity in the DNA damage than the ones bearing no such functionalities. Kinetic analysis by UV-visible spectra demonstrates that the formation of ArOH-Cu (II) complexes, the stabilization of oxidative intermediate derived from ArOH and Cu (II)/Cu (I) redox cycles, might be responsible for the DNA damage. This study also reveals a good correlation between antioxidant and prooxidant activity, as well as cytotoxicity against human leukemia (HL-60 and Jurkat) cell lines. The mechanisms and implications of these observations are discussed.     

Comparative genotoxicity of 3-hydroxyanthranilic acid and anthranilic acid in the presence of a metal cofactor Cu (II) in vitro

Several clinical studies have reported that an increase in excretion of tryptophan metabolites 3-hydroxyanthranilic acid (3-OHAA), anthranilic acid (AA) and other metabolites in the urine of bladder cancer patients are implicated to play a role in the etiology of bladder cancer; however the mechanisms involved are unknown. The present study compares the genotoxicity of tryptophan metabolites AA and 3-OHAA to cause mutagenesis in vitro. The DNA damage effects of tryptophan metabolites were analyzed using plasmid relaxation assay performed with AA and 3-OHAA at various concentrations between 50μM and 400μM in the presence of plasmid DNA pSP-72. Both AA and 3-OHAA did not show any plasmid relaxation activity when tested alone. However, 3-OHAA in the presence of metal cofactor Cu (II) induced plasmid relaxation by causing nicks in the plasmid. This effect was not observed in the presence of other metal cofactors Fe (II) and Mn (III). Cu (II) at increasing concentrations between 5μM and 20μM and in the presence of 100μM 3-OHAA showed an apparent dose-response in causing DNA strand breaks. The Cu (II) mediated mutagenic activation of 3-OHAA was further investigated using Ames Salmonella/microsome mutagenicity assay with reactive oxygen species (ROS) sensitive tester strain Salmonella TA102. When 100μg of 3-OHAA per plate was incubated with Cu (II) a significant increase in TA102 revertants was observed with an increase in the concentration of Cu (II) from 2.5μg to 50μg. In contrast, AA with Cu (II) at such low concentration was unable to cause any significant increase in number of the TA102 revertants. This evidence for mutagenicity with only 3-OHAA and Cu (II) but not AA suggests the presence of hydroxyl group at ortho position to amino group in 3-OHAA structurally, is critical in reacting with Cu (II) to generate genotoxicity.     

Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).     

Inactivation of Trypanosoma cruzi and Crithidia fasciculata topoisomerase I by Fenton systems

Fenton systems (H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 microM Fe(II) or Cu(II) with 3.0 mM H(2)O(2). Thiol compounds and free radical scavengers prevented Fenton system effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione (GSH), dithiothreitol (DTT), cysteine and N-acetyl-L-cysteine (NAC) entirely prevented the effect of the H(2)O(2)/Fe(II) system; mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, GSH, DTT and NAC protected 100%, cysteine, histidine and mannitol protected 28%, 34% and 48%, respectively, whereas ethanol was ineffective. With the H(2)O(2)/Cu(II) system and T. cruzi topoisomerase, DTT and histidine protected 100% and 60%, respectively, but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by the H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II) systems proved to be irreversible since it was not reversed by the more effective enzyme protectors. It is suggested that topoisomerases could act either as targets of 'reactive oxygen species' (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species in situ.     

DNA-binding and cleavage studies of novel copper(II) complex with L-phenylalaninate and 1,4,8,9-tetra-aza-triphenylene ligands

DNA-binding properties of novel copper(II) complex [Cu(l-Phe)(TATP)(H(2)O)](+), where L-Phe=L-phenylalaninate and TATP=1,4,8,9-tetra-aza-triphenylene are investigated using electronic absorption spectroscopy, fluorescence spectroscopy, voltammetry and viscosity measurement. It is found that the presence of calf thymus DNA results in a hypochromism and red shift in the electronic absorption, a quenching effect on fluorescence nature of ethidium bromide-DNA system, an enhanced response on voltammograms of [Co(phen)(3)](3+/2+)-DNA system, and an obvious change in viscosity of DNA. From absorption titration, fluorescence analysis and voltammetric measurement, the binding constant of the complex with DNA is calculated. The latter two methods reveal the stronger binding of [Cu(l-Phe)(TATP)(H(2)O)](+) complex to double strand DNA by the moderate intercalation than [Co(phen)(3)](3+). Such a binding induces the cleavage of plasmid pBR322 DNA in the presence of H(2)O(2).