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.     

Genotoxic effect of 6-gingerol on human hepatoma G2 cells

6-Gingerol, a major component of ginger, has antioxidant, anti-apoptotic, and anti-inflammatory activities. However, some dietary phytochemicals possess pro-oxidant effects as well, and the risk of adverse effects is increased by raising the use of doses. The aim of this study was to assess the genotoxic effects of 6-gingerol and to clarify the mechanisms, using human hepatoma G2 (HepG2) cells. Exposure of the cells to 6-gingerol caused significant increase of DNA migration in comet assay, increase of micronuclei frequencies at high concentrations at 20–80 and 20–40 μM, respectively. These results indicate that 6-gingerol caused DNA strand breaks and chromosome damage. To further elucidate the underlying mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH). In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-hydroxydeoxyguanosine (8-OHdG). Results showed that lysosomal membrane stability was reduced after treatment by 6-gingerol (20–80 μM) for 40 min, mitochondrial membrane potential decreased after treatment for 50 min, GSH and ROS levels were significantly increased after treatment for 60 min. These suggest 6-gingerol induces genotoxicity probably by oxidative stress; lysosomal and mitochondrial damage were observed in 6-gingerol-induced toxicity.     

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.     

DNA Damage Measured by the Comet Assay in Eight Agronomic Plants

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay — the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

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, 25–200 μM 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 50 μM 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 ≥50 μM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.     

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.     

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.     

Genotoxic risk and oxidative DNA damage in HepG2 cells exposed to perfluorooctanoic acid

Perfluorooctanoic acid (C8HF15O2, PFOA) is widely used in various industrial fields for decades and it is environmentally bioaccumulative. PFOA is known as a potent hepatocarcinogen in rodents. But it is not yet clear whether it is also carcinogenic in humans, and the genotoxic effects of PFOA on human cells have not yet been examined. In this study, the genotoxic potential of PFOA was investigated in human hepatoma HepG2 cells in culture using single cell gel electrophoresis (SCGE) assay and micronucleus (MN) assay. In order to clarify the underlying mechanism(s) we measured the intracellular generation of reactive oxygen species (ROS) using dichlorofluorescein diacetate as a fluorochrome. The level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) in PFOA-treated HepG2 cells. PFOA at 50-400 microM caused DNA strand breaks and at 100-400 microM MN in HepG2 cells both in a dose-dependent manner. Significantly increased levels of ROS and 8-OHdG were observed in these cells. We conclude that PFOA exerts genotoxic effects on HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS.     

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

Introduction: N-Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N-nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. Aims: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. Methods: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. Key results: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. Conclusions: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N-nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH > 13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague–Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3–6 and 22–26 h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20 mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology.Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40 min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.     

Comparison of DNA damage in human-derived hepatoma line (HepG2) exposed to the fifteen drinking water disinfection byproducts using the single cell gel electrophoresis assay

Disinfection of drinking water reduces pathogenic infection, but generates disinfection by-products (DBPs) in drinking water. In this study, the effect of fifteen DBPs on DNA damage in human-derived hepatoma line (HepG2) was investigated by the single cell gel electrophoresis (SCGE) assay. These fifteen DBPs are: four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and chloral hydrate (CH). Based on the minimal effective concentration (MEC) at which DBPs induced significant increase in olive tail moment (OTM), the rank order of DNA-damaging potency is: bromodichloromethane (BDCM)>dibromochloromethane (DBCM)>tribromomethane (TBM)>trichloromethane (TCM) of the four THMs; iodoacetic acid (IA)>bromoacetic acid (BA)>dibromoacetic acid (DBA)>dichloracetic acid (DCA)>trichloroacetic acid (TCA) of the five HAAs; dibromoacetonitrile (DBN)approximately dichloroacetonitrile (DCN)>trichloroacetonitrile (TCN) of the three HANs. The DNA damaging potency of MX and CH is similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Chloroacetic acid (CA) is not genotoxic in this assay. Our findings indicated that HepG2/SCGE is a sensitive tool to evaluate the genotoxicity of DBPs and iodinated DBPs are more genotoxic than brominated DBPs, but chlorinated DBPs are less genotoxic than brominated DBPs.     

Simultaneous determination of 8-hydroxy-2′-deoxyguanosine and 5-methyl-2′-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry

8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 5-methyl-2′-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40 pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02–100 ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70–7.47% for 8-OHdG and 1.07–7.06% for 5-mdC, respectively. The recoveries were 93.4–108.5% for 8-OHdG and 87.4–104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.     

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H₂O₂ is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, and more in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.     

A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3 cells

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a pediatric lysosomal storage disorder characterized by accumulation of autofluorescent storage material and neurodegeneration, which result from mutations in CLN3. The function of CLN3, a lysosomal membrane protein, is currently unknown. We report that CLN3 interacts with cytoskeleton-associated nonmuscle myosin-IIB. Both CLN3 and myosin-IIB are ubiquitously expressed, yet mutations in either produce dramatic consequences in the CNS such as neurodegeneration in JNCL patients and Cln3^−/− mouse models, or developmental deficiencies in Myh10^−/− mice, respectively. A scratch assay revealed a migration defect associated with Cln3^−/− cells. Inhibition of nonmuscle myosin-II with blebbistatin in WT cells resulted in a phenotype that mimics the Cln3^−/− migration defect. Moreover, inhibiting lysosome function by treating cells with chloroquine exacerbated the migration defect in Cln3^−/−. Cln3^−/− cells traversing a transwell filter under gradient trophic factor conditions displayed altered migration, further linking lysosomal function and cell migration. The myosin-IIB distribution in Cln3^−/− cells is elongated, indicating a cytoskeleton defect caused by the loss of CLN3. In summary, cells lacking CLN3 have defects that suggest altered myosin-IIB activity, supporting a functional and physical interaction between CLN3 and myosin-IIB. We propose that the migration defect in Cln3^−/− results, in part, from the loss of the CLN3–myosin-IIB interaction.     

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk mouse lymphoma cells

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk^+/− mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations ≥50 μM was reduced to <50%. In the dose range up to 25 μM, viability was >90%. Measures of comet-tail length and thymidine–kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 μM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at ≥100 μM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.     

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.     

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells

Aim of the present study was the investigation of the genotoxicity of amino-alpha-carboline (AalphaC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AalphaC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations > or =50 microM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AalphaC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 microM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at > or =50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AalphaC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AalphaC is higher than that of other HAs that are formed during the cooking of meat and fish.     

Chemical composition and antigenotoxic properties of Lippia alba essential oils

The present work evaluated the chemical composition and the DNA protective effect of the essential oils (EOs) from Lippia alba against bleomycin-induced genotoxicity. EO constituents were determined by Gas Chromatography/Mass Spectrometric (GC-MS) analysis. The major compounds encountered being citral (33% geranial and 25% neral), geraniol (7%) and trans-β-caryophyllene (7%) for L. alba specimen COL512077, and carvone (38%), limonene (33%) and bicyclosesquiphellandrene (8%) for the other, COL512078. The genotoxicity and antigenotoxicity of EO and the compounds citral, carvone and limonene, were assayed using the SOS Chromotest in Escherichia coli. The EOs were not genotoxic in the SOS chromotest, but one of the major compound (limonene) showed genotoxicity at doses between 97 and 1549 mM. Both EOs protected bacterial cells against bleomycin-induced genotoxicity. Antigenotoxicity in the two L. alba chemotypes was related to the major compounds, citral and carvone, respectively. The results were discussed in relation to the chemopreventive potential of L. alba EOs and its major compounds.