Chromium(III)-induced 8-hydroxydeoxyguanosine in DNA and its reduction by antioxidants: comparative effects of melatonin, ascorbate, and vitamin E.

Chromium compounds are well documented carcinogens. Cr(III) is more reactive than Cr(VI) toward DNA under in vitro conditions. In the present study, we investigated the ability of Cr(III) to induce oxidative DNA damage by examining the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA incubated with CrCl(3) plus H(2)O(2). We measured 8-OH-dG using HPLC with electrochemical detection. In the presence of H(2)O(2), we observed that Cr(III)-induced formation of 8-OH-dG in isolated DNA was dose and time dependent. Melatonin, ascorbate, and vitamin E (Trolox), all of which are free radical scavengers, markedly inhibited the formation of 8-OH-dG in a concentration-dependent manner. The concentration that reduced DNA damage by 50% was 0.51, 30.4, and 36.2 microM for melatonin, ascorbate, and Trolox, respectively. The results show that melatonin is 60- and 70-fold more effective than ascorbate or vitamin E, respectively, in reducing oxidative DNA damage in this in vitro model. These findings also are consistent with the conclusion that the carcinogenic mechanism of Cr(III) is possibly due to Cr(III)-mediated Fenton-type reactions and that melatonin's highly protective effects against Cr(III) relate, at least in part, to its direct hydroxyl radical scavenging ability.

Cr is found in the workplace primarily in the valence forms Cr(VI) and Cr(III) (11).
Cr(VI) compounds are more toxic and carcinogenic than Cr(III) (12,13) because Cr(VI), in contrast to Cr(III), can readily cross cellular membranes via nonspecific anion carriers (13)(14)(15). However, once inside cells, Cr(VI) is reduced through reactive Cr intermediates such as Cr(V) and Cr(IV) to the ultimate kinetically stable Cr(III) by cellular reductants including glutathione and vitamin C (13,16). Therefore, Cr(III) or other intermediate oxidation states probably play an important role in Cr(VI)-induced toxicity (16).
Cr(III), which was initially thought to be relatively nontoxic, recently was found to be more effective than Cr(VI) in causing genotoxicity in cell-free systems (11). Cr(III) interacts with DNA to induce DNA strand breaks, DNA-protein cross-links, and oxidative DNA base modifications such as the formation of 8-hydroxydeoxyguanosine (8-OH-dG) (17)(18)(19)(20)(21). 8-OH-dG is a key biomarker relevant to carcinogenesis because the formation of 8-OH-dG in DNA causes misincorporation during replication and subsequently leads to G->T transversions (22,23). The carcinogenic mechanisms of Cr(III) relate to its ability to generate hydroxyl radicals ('OH) from H202 via a Fentontype reaction (20,24). The highly toxic 'OH then targets DNA, resulting in oxidative DNA base adducts such as 8-OH-dG.
Melatonin, an indoleamine product of the pineal gland, is an endogenous 'OH scavenger and a highly effective antioxidant (25,26). In vitro melatonin is as effective or more effective than either glutathione and mannitol in reducing 'OH toxicity (25) and is possibly more efficient than vitamin E in reducing the toxicity of the peroxyl radical (27). Moreover, melatonin is highly lipophilic (28) as well as somewhat hydrophilic (29); therefore, it easily passes all known morphophysiologic barriers and enters all subcellular compartments. Melatonin has a high affinity for cell nuclei in mammalian tissues, where its concentration can be 5 times higher than levels found in blood (30). By measuring a variety of oxidative indexes (including levels of 8-OH-dG), earlier studies have shown that melatonin effectively protects DNA from oxidative damage induced by a number of free-radical-generating agents including safrole, kainic acid, lipopolysaccharide, ferric nitrilotriacetate, ischemia/reperfusion, and ionizing radiation both in vitro and in vivo (31)(32)(33)(34)(35)(36).
In the present study, we investigated the ability of melatonin to reduce Cr(III)induced oxidative DNA damage in vitro and compared melatonin's efficacy to that of two well-known antioxidants, vitamins E and C. We examined the formation of 8-OH-dG in calf thymus DNA with Cr3CI plus H202 using HPLC with electrochemical detection.

Materials and Methods
Reagents. We purchased calf thymus DNA, CrCl3e6H2O, H202, and ascorbate from Sigma (St. Louis, MO), and we obtained Trolox from Aldrich (Milwaukee, WI). Pure melatonin was a gift from Helssin Chemicals SA (Biasca, Switzerland). We purchased nuclease P1 and alkaline phosphatase from Boehringer Mannheim (Indianapolis, IN). We used MilliQ-purified H2O to prepare all solutions. All other chemicals were of the highest quality available. Treatment Assay for 8-OH-dG. After incubation, we added 50 1.L sodium acetate (3 M, pH 5.0) and two volumes of -200C to each sample to terminate the reaction. DNA was precipitated and washed once with 70% ethanol. The DNA sample was dried and dissolved in 200-pL 20 mM sodium acetate (pH 5.0); the samples were denatured by heating at 95°C for 5 min and then cooled on ice. The DNA samples were digested to nucleotides by incubation with 8 U nudease P1 at 37°C for 30 min. Next, we added 20 pL 1-M Tris-HCl (pH 8.0) to the samples and they were treated with 4 U alkaline phosphatase at 370C for 1 hr. We filtered the resulting deoxynudeoside mixture through a Millipore filter (0.22 pm; Millipore) and analyzed it using HPLC with an electrochemical detection system. We used an ESA HPLC system (ESA, Chelmsford, MA) equipped with an eight-channel CoulArray 5600 electrochemical detector: YMC-BD (4.6 mm x 250 mm, Partisil 5 p OD53; Waters, Milford, MA) column (3 pm, 150 x 4.6 mm i.d.). The eluent was a 10% aqueous methanol containing 12.5 mM citric acid, 25 mM sodium acetic acid, 30 mM sodium hydroxide, and 10 -mM acetic acid at a flow rate of 1 mL/min. We measured the quantities of 8-OH-dG and 2-deoxyguanosine (2-dG) using different channels and two oxidative potentials (300 and 900 mV, respectively). The level of 8-OH-dG in each sample was expressed as the ratio of 8-OH-dG to 105 2-dG (37). Statstical analysis. We analyzed all data by a one-way analysis of variance followed by the Tukey test.

Results
The levels of 8-OH-dG increased in a dosedependent manner with increasing concentrations of CrCl3 ( Figure 1). All concentrations of Cr(III) from 10 pM to 0.75 mM caused significant increases in 8-OH-dG levels in DNA. We selected a concentration of 0.5 mM Cr(III) for the subsequent studies because it yielded high levels of 8-OH-dG. In the second study, 8-OH-dG levels increased essentially in a linear manner during the incubation period when 0.5 mM CrCl3 plus 0.5 mM H202 were incubated with DNA ( Figure 2). We selected an intermediate time of 60 min for the subsequent studies because this incubation time produced optimal levels of 8-OH-dG. Figure 3 shows that melatonin inhibited Cr(III)-induced formation of 8-OH-dG in a dose-dependent manner. All melatonin concentrations > 0.25 pM significantly reduced 8-OH-dG formation in DNA induced by 0.5 mM Cr(III) plus 0.5 mM H202 (p < 0.05). Figure 4 shows that ascorbate inhibited Cr(III)-induced formation of 8-OH-dG in a dose-dependent manner. The effective concentrations of ascorbate against Cr(III)-induced formation of 8-OH-dG in DNA were between 1 and 250 pM. Figure 5 shows that the formation of 8-OH-dG in DNA was also inhibited by Trolox in a dosedependent manner. The effective concentrations of Trolox ranged from 10 to 250 pM. To compare the efficacy of melatonin, ascorbate, and Trolox, we calculated the percentage-inhibition curves (Figure 6). The IC50 is the concentration of a particular agent that inhibits the formation of 8-OH-dG in DNA by 50%. The IC50 for melatonin was 0.51 pM; this value is much less than for ascorbate (IC50 = 30.4 pM) or Trolox (IC50 = 36.2 pM).

Discussion
H202 is a normal metabolite in the cell; its steady-state concentrations range from 10-9 to 10-8 M (38). The concentrations of H202 may markedly increase in tissues when they are subjected to ionizing radiation, during the metabolism of carcinogens, and at sites of inflammation (39)(40)(41). Although H202 may not cause DNA damage under physiologic conditions, it participates in the metal ion-catalyzed Haber-Weiss reaction and generates the highly reactive 'OH, which can target DNA, resulting in oxidative DNA damage (42). Electron spin resonance spectroscopy studies have shown that 'OH are generated in a DNA-free solution containing Cr(III) and H202 (43). The present study demonstrates that Cr(III) plus H202 is capable of inducing oxidative DNA damage. When we incubated calf thymus DNA with CrCl3 + H202, the levels of 8-OH-dG detected were approximately 40   *p < 0.05 as compared to the Con group. than those in the untreated controls. Furthermore, the formation of 8-OH-dG increases in a dose-and time-dependent manner in the presence of 0.5 mM H202. Melatonin, ascorbate, and vitamin E (Trolox) all function as free radical scavengers and markedly inhibited the formation of 8-OH-dG in a concentration-dependent manner but, dearly, with different efficacies.
Vitamin E, a well-known antioxidant and inhibitor oflipid peroxidation in biologic membranes, has protective effects against the carcinogenic or mutagenic activity of chemical agents and ionizing radiation (16). We found that Trolox, a water-soluble vitamin E analogue, successfully inhibited the Cr(III)-induced formation of 8-OH-dG in isolated DNA in a concentration-dependent manner. Trolox concentrations > 10 mM significantly reduced 8-OH-dG levels. The IC50 value for Trolox was 36.2 pM.
In this in vitro system, we also found that ascorbate, a water-soluble physiologic antioxidant, had a protective effect, with an IC50 value of 30.4 pM calculated from its percentinhibition curve. Vitamin C has antiviral, anticancer, and antimutagenic activity (16). However, under certain conditions, vitamin C acts as prooxidant, generating free radicals (44). In a number of studies, ascorbate   (45)(46)(47). Thus, although ascorbate functions as an free radical scavenger in the Cr(III) plus H202 system, the utility of ascorbate in Cr detoxification in vivo should be cautiously considered.
As compared to ascorbate (IC50 = 30.4 gM) and vitamin E (IC50 = 36.2 PM), melatonin was more effective in reducing the formation of 8-OH-dG in this system (IC50 = 0.51 PM). Thus, melatonin was roughly 60-and 70-fold more effective in reducing oxidative damage to DNA than ascorbate and vitamin E, respectively. Also, the minimal concentration of melatonin required to significantly reduce 8-OH-dG formation was much less than that of either vitamin.  Results are given as means + SE (n= 5).
*p < 0.05 as compared to the Cr(IIt) plus H202 group. and is a particularly efficient scavenger of the highly toxic OH (25,48,49). Melatonin neutralizes two OH for each melatonin molecule, resulting in the formation of the product cyclic 3-hydroxymelatonin (50). In the present study, the formation of 8-OH-dG was thought to be due to a Cr(III)-mediated Fenton-type reaction that generates OH, which in turn attacked DNA, resulting in the accumulation of the oxidative DNA base adduct 8-OH-dG (20,24). Second, melatonin not only detoxifies the highly toxic 'OH, but also scavenges its precursor, H202. We recently uncovered a new pathway in which melatonin interacts with H202 to yield Nl-acetyl-N2-formyl-5methoxykynuramine (51). The structure of the product was confirmed using mass spectrometry, proton nuclear magnetic resonance, and carbon nuclear magnetic resonance. By lowering the concentration of H202, 'OH generation in this system would also be proportionally reduced. Such a dual strategy of antioxidant protection would be much more efficient than simply scavenging OH. Third, because melatonin is highly lipophilic (28) as well as somewhat hydrophilic (29, it easily enters cells and sub-cellular compartments. Intracellularly, the highest radioimmuoassayable concentrations of melatonin are measured in the nudei of brain cells after its peripheral administration to animals (30). Melatonin has a high affinity for the nucleus (and possibly DNA itself), which may contribute to its protective effect against Cr-induced formation of 8-OH-dG. Cr(III) accumulates in nuclei and has a high affinity for DNA (52). Melatonin may prevent the formation of 8-OH-dG by displacing Cr(III) from the Cr-DNA binding complex and thereby reduce H202-mediated 'OH generation in the vicinity of DNA. Fourth, melatonin and its precursors reportedly have a high metal-binding affinity (53). Limson et al. (53) showed that melatonin chelated aluminum, cadmium, iron, copper, and lead, etc. Although the authors did not investigate Cr, melatonin may also chelate this transition metal ion to prevent the formation of the 'OH via the Cr-mediated Fenton-type reaction: Cr(III) + H202 -Cr(IV) + 'OH + OH-. Susa et al. (54) used different end points and cultured primary rat hepatocytes and found that melatonin markedly reduced nuclear DNA single-strand breaks induced by K2Cr207 [Cr(VI)]. They speculated that melatonin protected cells from free radical toxicity by one of several means, including melatonin's ability to preserve intracellular levels of vitamins E and C, stimulation of catalase activity, and/or by directly scavenging the 'OH. In current studies, two of the options proposed by Susa et al. (54), i.e., maintenance of Vitamin E and C levels and the stimulation of catalase activity, were clearly not involved in melatonin's protection of DNA from oxidative damage. Thus, the most likely explanation for the current findings is that melatonin's effects were a consequence of its ability to scavenge the 'OH and possibly also H202.
In the current in vitro study, which used purified DNA, the curves for the inhibition of DNA damage by each of the three antioxidants, i.e., melatonin, ascorbate, and Trolox ( Figure 6), were quite different. The relevance of these curves to the pharmacologic utility of these molecules in protecting nuclear DNA from oxidative damage in vivo remains to be investigated. However, in in vivo studies where other free-radical-generating agents were used, melatonin also proved highly effective in reducing DNA damage consistent with its ability to enter the nucleus with ease (55,56). There have been no in vivo studies where vitamin E, ascorbate, and melatonin were compared for their relative efficacies in protecting DNA from oxidative destruction.
Melatonin as an antioxidant is effective in protecting membrane lipids, nuclear DNA, and protein from oxidative damage induced by a variety of free-radical-generating agents and processes both in vitro as well as in vivo (26,(55)(56)(57)(58). Considering the apparent virtual absence of acute or chronic toxicity, melatonin's clinical application against Crinduced genotoxicity in occupational and environmental situation where this metal is a problem should be considered.