Cytotoxicity and genotoxicity of methyleugenol and related congeners-- a mechanism of activation for methyleugenol

Methyleugenol is a substituted alkenylbenzene found in a variety of foods, products, and essential oils. In a 2-year bioassay conducted by the National Toxicology Program, methyleugenol caused neoplastic lesions in the livers of Fischer 344 rats and B6C3F(1) mice. We were interested in the cytotoxicity and genotoxicity caused by methyleugenol and other alkenylbenzene compounds: safrole (a known hepatocarcinogen), eugenol, and isoeugenol. The endpoints were evaluated in cultured primary hepatocytes isolated from male Fischer 344 rats and female B6C3F(1) mice. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release, while genotoxicity was determined by using the unscheduled DNA synthesis (UDS) assay. Rat and mouse hepatocytes showed similar patterns of toxicity for each chemical tested. Methyleugenol and safrole were relatively non-cytotoxic, but caused UDS at concentrations between 10 and 500 microM. In contrast, isoeugenol and eugenol produced cytotoxicity in hepatocytes with LC50s of approximately 200-300 microM, but did not cause UDS. Concurrent incubation of 2000 microM cyclohexane oxide (CHO), an epoxide hydrolase competitor, with a non-cytotoxic concentration of methyleugenol (10 microM) resulted in increased cytotoxicity but had no effect on genotoxicity. However, incubation of 15 microM pentacholorophenol, a sulfotransferase inhibitor, with 10 uM methyleugenol resulted in increased cytotoxicity but had a significant reduction of genotoxicity. These results suggest that methyleugenol is similar to safrole in its ability to cause cytotoxicity and genotoxicity in rodents. It appears that the bioactivation of methyleugenol to a DNA reactive electrophile is mediated by a sulfotransferase in rodents, but epoxide formation is not responsible for the observed genotoxicity.     

Hydroquinone-induced genotoxicity and oxidative DNA damage in HepG2 cells

Hydroquinone (HQ) is used as an antioxidant in rubber industry and as a developing agent in photography. HQ is also an intermediate in the manufacture of rubber, food antioxidant and monomer inhibitor. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of HQ and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. DNA strand breaks and DNA-protein crosslinks (DPC) were measured by the proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. Using the SCGE assay, a significant dose-dependent increment in DNA migration was detected at concentrations of HQ (6.25-25 microM); but at the higher tested concentrations (50 microM), a reduction in the migration compared to the maximum migration at 25 microM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of HQ (50 microM). A significant increase of the frequency of micronuclei was found in the range from 12.5 to 50 microM in the micronucleus test (MNT). The data suggested that HQ caused DNA strand breaks, DPC and chromosome breaks. To elucidate the oxidative DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were chosen to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that HQ induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 25-50 and 6.25-50 microM, respectively. Moreover, HQ significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 12.5 to 50 microM. All these results demonstrate that HQ exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress. GSH, as a main intracellular antioxidant, is responsible for cellular defense against HQ-induced DNA damage.     

Free radical reactions of a naturally occurring flavone baicalein and possible mechanisms towards its membrane protective properties

Baicalein (5, 6, 7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavone present in some of the medicinal plants is known for its potential therapeutic effects, such as cardioprotective, anticancer and anti-inflammatory properties. However, detailed role and mechanisms behind its protective properties against different generators for oxidative stress have not been examined. In the present study, we investigated the possible protective ability of baicalein against the membrane damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the mechanisms involved using pulse radiolysis technique. Baicalein offered efficient protection even at a concentration of 10 microM towards membrane damage caused by lipid peroxidation induced by the gamma-radiation, peroxyl radicals, ascorbate-Fe2+ and peroxynitrite in rat liver mitochondria and heart homogenate. To elucidate its reaction mechanisms with biologically relevant radicals, transient absorption spectroscopy employing pulse radiolysis technique was used. Baicalein showed fairly high rate constants (3.7 x 10(9), 1.3 x 10(9) and 8.0 x 10(8) dm3 mol(-1) s(-1) for hydroxyl, azidyl and alkylchloroperoxyl radicals, respectively), suggesting that baicalein can act as an effective scavenger of these radicals. In each case, the phenoxyl radical of baicalein was generated. Thus, it was evident that the phenolic moiety of baicalein was responsible for the free radical scavenging process. Baicalein also reacts with linoleic acid peroxyl radical (LOO*), indicating its ability to act as a chain breaking antioxidant. Peroxynitrite-mediated radicals were shown to be reactive towards baicalein and the bimolecular rate constants were 2.5 x 10(7) and 3 x 10(8) dm3 mol(-1) s(-1) for *NO2 and CO3*(-) radicals, respectively. In conclusion, our results revealed the potential of baicalein in protecting mitochondrial membrane against oxidative damage induced by the four different agents. We propose that the protective effect is mediated via scavenging of primary and secondary radicals generated during oxidative stress.     

Hemoglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes

Epidemiological findings have indicated that red meat increases the likelihood of colorectal cancer. Aim of this study was to investigate whether hemoglobin, or its prosthetic group heme, in red meat, is a genotoxic risk factor for cancer. Human colon tumor cells (HT29 clone 19A) and primary colonocytes were incubated with hemoglobin/hemin and DNA damage was investigated using the comet assay. Cell number, membrane damage, and metabolic activity were measured as parameters of cytotoxicity in both cell types. Effects on cell growth were determined using HT29 clone 19A cells. HT29 clone 19A cells were also used to explore possible pro-oxidative effects of hydrogen peroxide (H2O2) and antigenotoxic effects of the radical scavenger dimethyl sulfoxide (DMSO). Additionally we determined in HT29 clone 19A cells intracellular iron levels after incubation with hemoglobin/hemin. We found that hemoglobin increased DNA damage in primary cells (> or =10 microM) and in HT29 clone 19A cells (> or =250 microM). Hemin was genotoxic in both cell types (500-1000 microM) with concomitant cytotoxicity, detected as membrane damage. In both cell types, hemoglobin and hemin (> or =100 microM) impaired metabolic activity. The growth of HT29 clone 19A cells was reduced by 50 microM hemoglobin and 10 microM hemin, indicating cytotoxicity at genotoxic concentrations. Hemoglobin or hemin did not enhance the genotoxic activity of H2O2 in HT29 clone 19A cells. On the contrary, DMSO reduced the genotoxicity of hemoglobin, which indicated that free radicals were scavenged by DMSO. Intracellular iron increased in hemoglobin/hemin treated HT29 clone 19A cells, reflecting a 40-50% iron uptake for each compound. In conclusion, our studies show that hemoglobin is genotoxic in human colon cells, and that this is associated with free radical mechanisms and with cytotoxicity, especially for hemin. Thus, hemoglobin/hemin, whether available from red meat or from bowel bleeding, may pose genotoxic and cytotoxic risks to human colon cells, both of which contribute to initiation and progression of colorectal carcinogenesis.     

Induction of micronuclei in cultured murine splenocytes exposed to elevated levels of ferrous ions, hydrogen peroxide and ultraviolet irradiation

The frequency of micronuclei in cultured mouse splenocytes increased positively and in a dose-related manner to exposure to ferrous ions and ultraviolet irradiation, but not to hydrogen peroxide. Combined treatments, especially when ferrous ions were present with hydrogen peroxide or with ultraviolet irradiation, led to a synergistic enhancement in micronucleus frequency. The results indicate that a significant level of chromosome damage is associated with exposure to ultraviolet light and to general cellular pro-oxidative stress, and indicate that under these conditions the micronucleus assay can provide an effective in vitro model for the study of genotoxicity in relation to oxygen-derived free radicals.     

Inhibition of acrylamide genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol

The chemoprotective effect of hydroxytyrosol (HT), a strong antioxidant compound from extra virgin olive oil, against acrylamide (AA)-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The micronucleus test (MNT) assay was used to monitor genotoxicity. In MNT, we found that HT at all tested concentrations (12.5-50 microM) significantly reduced the micronuclei frequencies in a concentration-dependent manner caused by AA. In order to clarify the underlying mechanisms we measured the intracellular reactive oxygen species (ROS) formation using 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS) and gamma-GCS was measured using Western blotting. The results showed that HT significantly concentration-dependent reduced the genotoxicity caused by AA. Furthermore, HT was able to reduce intracellular ROS formation and attenuate GSH depletion caused by AA in a concentration-dependent manner. It was also found that HT enhanced the expression of gamma-GCS in HepG2 cells treated with 10 mM AA using immunoblotting in a concentration-dependent manner. The results showed that HT reduced the AA-induced genotoxicity by decreasing the ROS level and increasing the GSH level. The data strongly suggest that HT have significant protective ability against AA-induced genotoxicity in vitro.     

Influence of antimonite, selenite, and mercury on the toxicity of arsenite in primary rat hepatocytes

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.     

Genotoxicity of HNE

Since previous investigations on the genotoxicity of 4-hydroxynonenal (HNE) were carried out with prokaryotic systems or eukaryotic cell lines which may not adequately reflect the response of cells in vivo due to differences in the metabolism, the genotoxic potential of HNE was further evaluated in primary cells (hepatocytes) and cell clones of cerebral endothelial cells expressing specific functions, i.e. blood-brain barrier (BBB) and capillary formation associated phenotypes. Treatment of hepatocytes with HNE induced statistically significant levels of SCE at concentrations >/=0.1 microM, micronuclei at concentrations >/=1 microM and chromosomal aberrations at a concentration of 10 microM. Treatment of cloned cerebral microvascular endothelial cells induced significantly elevated levels of chromosomal aberrations at concentrations >/=1 microM and micronuclei at concentrations >/=10 microM in both cEC phenotypes, compared to the controls. Additionally, cytotoxicity was observed at a concentration of 50 microM HNE and was significantly higher in type II cells. These results indicate that cells expressing differentiated functions representative for the in vivo situation react more sensitive to HNE than cell lines, and may reflect the sensitivity of the target cells. The different response with respect to the endpoints of genotoxicity tested most probably depends on the different metabolizing capacities and thus the action of different metabolites of HNE.     

The in vitro micronucleus technique

The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.     

Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells

Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identify and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25-100 microM) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50-100 and 50-100 microM, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses.     

Reactions of oxyl radicals with DNA

The importance of radical-induced damage to DNA is apparent from the ever-increasing number of publications in this area. This review focuses on the damage caused to DNA by reactive oxygen-centred radicals, however formed. These may be hydroxyl radicals, which arise either from the radiolysis of water by ionizing radiation (γ-rays or X-rays), or from a purely chemical source. Alternatively, metal-bound oxyl radicals (M–O·) are also active intermediates in DNA-cleaving reactions and may be formed from synthetic compounds or from natural products such as bleomycin (BLM). Chemical mechanisms leading to the observed degradation products are covered in detail. The biological effects of some of the DNA base lesions formed are touched upon, concentrating on the molecular mechanisms behind the initial events that lead to mutagenesis.     

Possible mutagens derived from lipids and lipid precursors

Free radicals can initiate the oxidative decomposition of cellular membranes by lipid peroxidation. In this process a great variety of reactive aldehydes are produced intracellularly. Some of them, such as 4-hydroxynonenal or malonaldehyde, are biologically very active and might be involved in free radical-mediated DNA damage. A short review of the effects of aldehydic lipid peroxidation products on isolated DNA, their genotoxic effect in prokaryotes and eukaryotes and their in vivo carcinogenicity is given. Additionally own experiments on cytotoxic and genotoxic effects of 4-hydroxynonenal, 2-nonenal and nonanal in primary cultures of rat hepatocytes are reported. 4-Hydroxynonenal was highly cytotoxic at 100 microM, at subcytotoxic concentrations of 0.1-10 microM 4-hydroxynonenal increased the frequency of micronuclei, chromosomal aberrations and sister-chromatid exchange. 2-Nonenal and nonanal were not cytotoxic at 100 microM, the maximum dose tested. At 100 microM 2-nonenal led to a slight increase in micronuclei; chromosomal aberrations were not significantly altered. Nonanal had no detectable genotoxic effects. The level of endogenous 4-hydroxynonenal in tissues is in the range of 0.1-3.0 microM and can increase to 10 microM in conditions of oxidative stress; such levels appear to be sufficiently high to produce DNA damages, whether such damages are transient or irreversible is not known.     

Study of the biological effects and DNA damage exerted by a new dipalladium-Hmtpo complex on human cancer cells

The new dipalladium complex [Pd(2)(mu-mtpo-N(3),N(4))(2)(phen)(2)](NO(3))(2) (where phen=1,10-phenantroline; Hmtpo=5,7-dihydro-7-oxo-5-methyl[1,2,4]triazolopyrimidine), (Pd(2)-Hmtpo, or complex I), interacts effectively with DNA plasmid (pBS), as studied by circular dichroism spectroscopy (CD), causing large helix distortions, altering the direction of the main DNA helix axis and producing unwinding of the DNA double helix. DNA damage induced by complex I was highly significant at 2.81 microM (ovarian carcinoma TG cell line), as assessed by comet assay, a dose at which all treated nuclei showed more than 30% DNA migration to the comet tail. DNA damage effect is a consequence of genotoxicity and not a false positive response caused by cytotoxicity. In vitro cytotoxic assay on the two human tumor cell lines TG and BT-20 (breast carcinoma), shows that doses of 0.47, 1.41 and 2.81 microM produce significant antiproliferative effects after 4 days of treatment compared with control. Complex I was highly cytotoxic at 2.81 microM causing an inhibition of viable cells of 65.5%. Cisplatin (cis-DDP) exhibits lower cytotoxic activity in TG cells than dipalladium complex (a cisplatin dose of 6.67 microM inhibits 30.3%) and does not cause migration of DNA to comet tail.     

Possible involvement of oxidative stress in trichloroethylene-induced genotoxicity in human HepG2 cells

Trichloroethylene (TCE) is an environmental and industrial pollutant whose hepatotoxicity has been demonstrated in experimental animals. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of TCE and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. Exposure of the cells to TCE caused significant increase of DNA migration in comet assay and of micronuclei (MN) frequencies at all tested concentrations (0.5-4mM), respectively, which suggests that TCE caused DNA strand breaks and chromosome damage. The involvement of lipid peroxidation in the genotoxic properties of TCE was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). To elucidate the role of glutathione (GSH) in these effects, the intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by co-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to TCE-induced cytotoxicity and DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by TCE was almost completely prevented. These results indicate that TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.     

Effects of scavengers of reactive oxygen and radical species on cell survival following photodynamic treatment in vitro: comparison to ionizing radiation

The effects of various scavengers of reactive oxygen and/or radical species on cell survival in vitro of EMT6 and CHO cells following photodynamic therapy (PDT) or gamma irradiation were compared. None of the agents used exhibited major direct cytotoxicity. Likewise, none interfered with cellular porphyrin uptake, and none except tryptophan altered singlet oxygen production during porphyrin illumination. The radioprotector cysteamine (MEA) was equally effective in reducing cell damage in both modalities. In part, this protection seems to have been induced by oxygen consumption in the system due to MEA autoxidation under formation of H2O2. The addition of catalase, which prevents H2O2 buildup, reduced the effect of MEA to the same extent in both treatments. Whether the remaining protection was due to MEA's radical-reducing action or some remaining oxygen limitation is unclear. The protective action of MEA was not mediated by a doubling of cellular glutathione levels, since addition of buthionine sulfoximine, which prevented glutathione increase, did not diminish the observed MEA protection. The hydroxyl radical scavenger mannitol also afforded protection in both kinds of treatment, but it was approximately twice as effective in gamma irradiation as in PDT. This is consistent with the predominant role of OH radicals in ionizing radiation damage and their presumed minor involvement in PDT damage. Superoxide dismutase, a scavenger of O2, acted as a radiation protector but was not significantly effective in PDT. Catalase, which scavenges H2O2, was ineffective in both modalities. Tryptophan, an efficient singlet oxygen scavenger, reduced cell death through PDT by several orders of magnitude while being totally ineffective in gamma irradiation. These data reaffirm the predominant role of 1O2 in the photodynamic cell killing but also indicate some involvement of free radical species.     

The genotoxic potential of glutaraldehyde in mammalian cells in vitro in comparison with formaldehyde

Glutaraldehyde (GA) induces DNA-protein crosslinks (DPX), but conflicting results have been reported with regard to other genotoxic and mutagenic effects in mammalian cells in vitro. We, therefore, characterized the genotoxic and mutagenic potential of GA in V79 cells. Using the alkaline comet assay we demonstrated the induction of DPX by GA (reduction of gamma ray-induced DNA migration) at a concentration of 10 microM and above. The standard comet assay did not reveal a significant DNA strand-breaking activity of GA. Cross-linking concentrations of GA were also cytotoxic, i.e. inhibited cell growth of treated V79 cultures. Interestingly, a small but statistically significant increase in sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (2 and 5 microM). FISH analysis revealed that the majority of GA-induced MN was due to chromosome breaks. We also compared the genotoxic activity of GA to that of formaldehyde (FA). Similar to GA, FA-induced DPX, SCE and MN, but distinct differences exist with regard to the sensitivity of the endpoints and the relationship between genotoxicity and cytotoxicity. However, the differences in genotoxicity cannot readily explain the different carcinogenic activities of the two compounds.     

Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies

Phenolphthalein induces tumors in rodents but because it is negative in assays for mutation in Salmonella and in mammalian cells, for DNA adducts and for DNA strand breaks, its primary mechanism does not seem to be DNA damage. Chromosome aberration (Ab) induction by phenolphthalein in vitro is associated with marked cytotoxicity. At very high doses, phenolphthalein induces weak increases in micronuclei (MN) in mouse bone marrow; a larger response is seen with chronic treatment. All this suggests genotoxicity is a secondary effect that may not occur at lower doses. In heterozygous TSG-p53((R)) mice, phenolphthalein induces lymphomas and also MN, many with kinetochores (K), implying chromosome loss. Induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas.Here we address some of the postulated mechanisms of genotoxicity in vitro, including metabolic activation, inhibition of thymidylate synthetase, cytotoxicity, oxidative stress, DNA damage and aneuploidy. We show clearly that phenolphthalein does not require metabolic activation by S9 to induce Abs. Inhibition of thymidylate synthetase is an unlikely mechanism, since thymidine did not prevent Ab induction by phenolphthalein. Phenolphthalein dramatically inhibited DNA synthesis, in common with many non-DNA reactive chemicals that induce Abs at cytotoxic doses. Phenolphthalein strongly enhances levels of intracellular oxygen radicals (ROS). The radical scavenger DMSO suppresses phenolphthalein-induced toxicity and Abs whereas H(2)O(2) potentiates them, suggesting a role for peroxidative activation. Phenolphthalein did not produce DNA strand breaks in rat hepatocytes or DNA adducts in Chinese hamster ovary (CHO) cells. All the evidence points to an indirect mechanism for Abs that is unlikely to operate at low doses of phenolphthalein. We also found that phenolphthalein induces mitotic abnormalities and MN with kinetochores in vitro. These are also enhanced by H(2)O(2) and suppressed by DMSO. Our findings suggest that induction of Abs in vitro is a high-dose effect in oxidatively stressed cells and may thus have a threshold. There may be more than one mechanism operating in vitro and in vivo, possibly indirect genotoxicity at high doses and also chromosome loss, both of which would likely have a threshold.     

Genotoxic effect and nitrative DNA damage in HepG2 cells exposed to aristolochic acid

Aristolochic acid (AA), extensively used as a traditional herbal medicine, was withdrawn from the market in the last century because it was found to be a potent carcinogen in humans and animals. The aim of this study was to evaluate the genotoxic effect of AA and obtain further insight into whether the nitrative DNA damage can be induced by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivative peroxynitrite (ONOO(-)) using human hepatoma HepG2 cells. To identify the genotoxic effect, the comet assay and micronucleus test (MNT) were performed. In the comet assay, 25-200microM of AA caused a significant increase of DNA migration in a dose-dependent manner. A significant increase of the frequency of micronuclei was found in the range between 12.5 and 50microM in the MNT. The results showed that AA caused DNA and chromosome damages. To elucidate the nitrative DNA damage mechanism, the level of nitrite and 8-hydroxydeoxyguanosine (8-OHdG), which can be generated by ONOO(-), were monitored with the 2,3-diaminonaphthalene (DAN) assay and immunoperoxidase staining, respectively. The results showed that AA causes a significant increase in the levels of NO and formation of 8-OHdG at concentrations >/=50microM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.     

Enhancement of DNA damage and involvement of reactive oxygen species after exposure to bitumen with UVA irradiation.

This study was carried out to evaluate whether bitumen cytotoxicity is enhanced when bitumen treatment is combined with UVA exposure. We also evaluated the oxidative processes in bitumen-induced DNA damage, and attempted to identify the DNA damage caused by bitumen and UVA exposures, either alone or in combination. The effects of bitumen and UVA on cell proliferation were examined using HL 60 cells. DNA-protein crosslinks (DPCs) were assessed using a K-SDS assay, and reactive oxygen species formation was detected by 8-OH-dG formation. We evaluated the formations of double-strand breaks (DSB) using lambdaDNA/HindIII and single-strand breaks (SSB) using PM2 DNA. The cytotoxicity assay showed enhanced suppression of cell proliferation when bitumen exposure and UVA exposure were combined. Combined exposure caused significant increases in DPCs over either exposure alone. Incubation of deoxyguanosine (dG) with bitumen or UVA showed an increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels when compared with controls, and combined exposure enhanced this effect. An evaluation of agarose gel bands showed that DSB and SSB were not formed following exposure to bitumen and UVA. This fact indicates that bitumen and UVA may be involved in genotoxic processes by producing oxygen free radicals and that combined exposure enhances these effects.     

Possible involvement of oxidative stress in potassium bromate-induced genotoxicity in human HepG2 cells

Potassium bromate (KBrO₃, PB) is a by-product of ozone used as disinfectant in drinking water. And PB is also a widely used food additive. However, there is little known about its adverse effects, in particular those related to its genotoxicity in humans. The aim of this study was to investigate the genotoxic effects of PB and the underlying mechanisms, using human hepatoma cell line, HepG2. Exposure of the cells to PB caused a significant increase of DNA migration in single cell gel electrophoresis (SCGE) assay and micronuclei (MN) frequencies in micronucleus test (MNT) at all tested concentrations (1.56–12.5 mM and 0.12–1 mM), which suggested that PB-mediated DNA strand breaks and chromosome damage. To indicate the role of antioxidant in those effects, DNA migration was monitored by pre-treatment with hydroxytyrosol (HT) as an antioxidant in SCGE assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. To elucidate the genotoxicity mechanisms, the study monitored the levels of reactive oxygen species (ROS), glutathione (GSH) and 8-hydroxydeoxyguanosine (8-OHdG). PB was shown to induce ROS production (12.5 mM), GSH depletion (1.56–12.5 mM) and 8-OHdG formation (6.25–12.5 mM) in HepG2 cells. Moreover, lysosomal membrane stability and mitochondrial membrane potential were further studied for the mechanisms of PB-induced genotoxicity. A significant increase was found in the range of 6.25–12.5 mM in lysosomal membrane stability assay. However, under these PB concentrations, we were not able to detect the changes of mitochondrial membrane potential. These results suggest that PB exerts oxidative stress and genotoxic effects in HepG2 cells, possibly through the mechanisms of lysosomal damage, an earlier event preceding the oxidative DNA damage.