Alkaline Single-cell Microgel Electrophoresis Research Articles

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro

Background aimsAdipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. MethodsAfter isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. ResultsDuring in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. ConclusionsThe study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell–based therapy.

Chlorophyll derivative mediated PDT versus methotrexate: An in vitro study using MCF-7 cells

Breast cancer is the most common cause of cancer deaths among women worldwide. Although chemotherapy is a standard method for the treatment of breast cancer, the photodynamic therapy (PDT) is a recent promising modality for cancer diagnosis and treatment. Its major advantages over chemotherapy are better selectivity of tumour tissue destruction and lack of severe local and systemic complications. This work is directed towards evaluation of the efficacy of Photodynamic therapy using chlorophyll derivative (CHL) as a photosensitizer in treatment of breast cancer. It also aims at investigation of the genetic safety of chlorophyll mediated PDT in comparison to the conventional chemotherapy. Both methotrexate (MTX) and light activated chlorophyll derivative were used to target MCF-7 breast cancer cell line. Standard karyotyping and alkaline single cell microgel electrophoresis assay (Comet assay) were applied on normal human peripheral blood lymphocytes (HPL) in order to investigate the respective possible mutagenic and genotoxic side effects that might result from application of each therapeutic modality. Results obtained from this study showed that 50% of MCF-7 tumour cell death (LC(50)) was reached by using a concentration of chlorophyll derivative that is 138 times lower than MTX. Moreover, chlorophyll derivative exerted no genetic side effects as compared to MTX that resulted into several types of chromosomal breakages. Compared to MTX, light activated chlorophyll derivative proved to be a better candidate for breast cancer cell toxicity, referring to its higher efficacy at tumour cells killing, safety to normal cells and simple method of extraction.

Ex vivo toxicity of nitrogen dioxide in human nasal epithelium at the WHO defined 1-h limit value

Current pollution limits indicating potential harm to human health caused by nitrogen dioxide have prompted a variety of studies on the cytotoxicity and genotoxicity of nitrogen dioxide (NO 2) in vitro. The present study focuses on toxic effects of NO 2 at the WHO defined 1-h limit value of 200 μg NO 2/m 3 air, equivalent to 0.1 ppm NO 2. Nasal epithelial mucosa cells of 10 patients were cultured as an air–liquid interface and exposed to 0.1 ppm NO 2 for 0.5 h, 1 h, 2 h and 3 h and synthetic air as negative control. After exposure, analysis of genotoxicity was performed by the alkaline single cell microgel electrophoresis (comet) assay and by the micronucleus test. Depression of proliferation and cytotoxic effects were checked by the micronucleus assay and the trypan blue exclusion assay. The experiments demonstrated significant DNA fragmentation even at the shortest exposure duration of half an hour in the comet assay. The amount of DNA fragmentation significantly increased with extended NO 2 exposure durations. The amount of DNA fragmentation increased with extended exposure durations to synthetic air at a significantly lower level as compared to NO 2 exposure. Micronucleus inductions were seen only at the longest exposure duration of 3 h. There were no changes in proliferation seen in the micronucleus assay under any experimental setup. Moreover, no signs of necrosis, apoptosis or changes in viability were detected. Data demonstrate genotoxicity of NO 2 at concentrations found in the urban atmosphere during short exposure durations. DNA alterations in the micronucleus assay at an exposure time of 3 h indicate a significant DNA alteration possibly being hazardous to humans.

Cartilage tissue engineering for auricular reconstruction: In vitro evaluation of potential genotoxic and cytotoxic effects of scaffold materials

Tissue engineering of autologous cartilage transplants is suggested as a new approach in reconstruction of external auricular deformities. 1.6-Hexanediol (HD), 1.8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 6-hydroxyhexanoic acid (HHA) are matrices of the open-pored polyurethane three-dimensional scaffold. Since these bioresorbable materials may interact with the human organism, cytotoxic effects on human chondrocytes and lymphocytes and genotoxic effects on human lymphocytes were monitored. Staining with propidium iodide and fluorescence diacetate as well as the EZ4U proliferation assay served for the detection of cytotoxic effects of the materials on human chondrocytes. Trypan blue staining was used to monitor cytotoxicity on lymphocytes. Genotoxic effects on lymphocytes in terms of strand breaks, alkali labile sites and incomplete excision repair were determined by the alkaline single cell microgel electrophoresis (Comet) assay. Cytotoxic effects in chondrocytes and lymphocytes as well as genotoxic effects in lymphocytes were dose-dependent with threshold values of 5 mg/mL HD, 0.5 mg/mL DBU and 0.03 mg/mL HHA showing no effects. These data suggest that these matrices could be safely used for scaffolds made of polyurethane unless these compounds are not released at a rate giving higher concentrations at the site of implantation or in body fluids, respectively.

Cytotoxic and genotoxic effects of matrices for cartilage tissue engineering

Customizing auricles with biodegradable polyurethane colonized with autologous chondrocytes as an approach for tissue engineering cartilage transplants has been suggested for the reconstruction of the external ear to repair auricular deformities. Dextrose, triethanolamine and poly(ethylene glycol)- block-poly(propylene glycol)- block-poly(ethylene glycol) (PEG-PPG-PEG) are matrices of an open-pored polyurethane three-dimensional scaffold. After release from the polymer, these compounds can be absorbed into the human organism. Therefore, cytotoxic effects on human chondrocytes and lymphocytes and genotoxic effects on human lymphocytes were determined. Propidium iodide and fluoresceine diacetate staining as well as quantitative proliferations testing with EZ4U served to detect cytotoxic effects on chondrocytes. In lymphocytes cytotoxicity was checked by trypan blue staining and the alkaline single cell microgel electrophoresis (Comet) assay was used to study genotoxic effects. Dose-dependent cytotoxicity and genotoxicity of the matrices could be shown. Concentrations up to 4.25 mg/ml for dextrose, 0.15 mg/ml for PEG-PPG-PEG and 0.9 mg/ml for triethanolamine did not show cytotoxic effects in chondrocytes or genotoxic effects in lymphocytes. These data suggest that dextrose, triethanolamine and PEG-PPG-PEG could be safely used if scaffolds made of open-pored polyurethane do not release these compounds at a rate giving higher concentrations at the site of implantation or in body fluids, respectively.

Genotoxicity of nicotine in cell culture of Caenorhabditis elegans evaluated by the comet assay

To assess the genotoxicity of nicotine, its DNA-damaging effect on Caenorhabditis elegans cells was tested with the alkaline single-cell microgel electrophoresis (comet) assay. The degree of DNA migration (a measure of possible DNA single-strand breaks, alkali-labile sites, and incomplete excision repair sites) was expressed as the head DNA%, tail length, and Olive tail moment. Large differences were found between experimental variants: 0, 1, 10, and 100 μM (-)-nicotine. At concentrations of 1 and 10 μM, no damages were detected by the comet assay, and the Olive tail moment and tail length were significantly lower than in the control (P < 0.001). The highest head DNA% and the lowest tail length and Olive tail moment were observed in the presence of 1 μM of nicotine. At 100 μM of nicotine, a significant increase (P < 0.001) was observed in Olive tail moment and tail length (up to 2.7- and 3-fold, respectively, compared to the control). The results are consistent with the lowest head DNA% among the three tested variants. This study demonstrated that nicotine treatment had dose-dependent effects on the level of DNA damage. Generally, a high dose of nicotine (100 μM) is genotoxic, while a reasonably low concentration has a protective effect. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine in C. elegans is discussed.

Epigallocatechin-3-gallate reduces DNA damage induced by benzo[a]pyrene diol epoxide and cigarette smoke condensate in human mucosa tissue cultures

Although epidemiological studies indicate cancer preventive effects of diets rich in fruit and vegetables, large clinical intervention studies conducted to evaluate dietary supplementation with micronutrients, mostly vitamins, showed disappointing results in large parts. In contrast, there is encouraging epidemiologic data indicating great chemopreventive potential of a large group of phytochemicals, namely polyphenols. This study shows the DNA protective effect epigallocatechin-3-gallate, a tea catechin, and one of the best-studied substances within this group, on carcinogen-induced DNA fragmentation in upper aerodigestive tract cells. Cell cultures from fresh oropharyngeal mucosa biopsies were preincubated with epigallocatechin-3-gallate in different concentrations before DNA damage was introduced with the metabolically activated carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide or cigarette smoke condensate. Effects on resulting DNA fragmentation were measured using the alkaline single-cell microgel electrophoresis (comet assay). Epigallocatechin-3-gallate significantly reduced benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced DNA damage by up to 51% (P<0.001). Fragmentation induced by cigarette smoke condensate could be lowered by 47% (P<0.001). Data suggest a cancer preventive potential of epigallocatechin-3-gallate as demonstrated on a subcellular level. An additional mechanism of tea catechin action is revealed by using a primary mucosa culture model.

Chemopreventive action of dexamethasone and α-tocopherol in oxidative stressed cells

Introduction: Recent research indicates a close connection of inflammation and cancer as presumed by Virchow in 1893. The growing understanding of cellular signalling and regulatory pathways reveals multiple links between inflammation and cancer. This study was designed to evaluate the influence of the anti-inflammatory drug dexamethasone and the antioxidant α-tocopherol on oxidative induced DNA damage, a major factor in the development of malignancies. Material and methods: Miniorgan cultures (MOC) of fresh biopsied human nasal mucosa were used to keep cells in their microenvironment and thus to mimic in vivo conditions. MOC were pretreated with dexamethasone and α-tocopherol in different concentrations on 1 or on 5 days before oxidative DNA damage was introduced by hydrogen peroxide. The effect of these substances on DNA damage was evaluated using the alkaline single cell microgel electrophoresis (Comet Assay). Results: Dexamethasone induced slight, but considerable DNA fragmentation by itself. It effectively protected cells from hydrogen peroxide induced DNA damage, leading to a maximum decrease of about 45% when preincubated on 5 days at 20 μM. α-Tocopherol most effectively reduced oxidative DNA fragmentation by about 38% when MOC were pretreated 5 days at 20 μM. Discussion: Our experimental data clearly shows the DNA protective action of dexamethasone and α-tocopherol with regard to oxidatively induced DNA damage, a major pathogenetic factor that inflammation and cancer have in common.

DNA-protektives Potential von Polyphenolen in humanen Schleimhautzellkulturen

The concept of field cancerization in the head and neck offers an excellent basis for chemopreventive interventions. Within the last few years, polyphenols, the most abundant phytochemicals in our diet, have been identified as interesting chemopreventive agents based on their multiple actions. This study was designed to add more experimental data regarding the chemopreventive features of epigallocatechin gallate (EGCG) and tannin (TA) in cultures of fresh biopsied tissue to epidemiologic studies and animal and cell line experiments. Miniorgan cultures (MOC) were produced from oropharyngeal mucosa-cubes about 1 mm(3), epithelialized and with their tissue structure preserved. The MOC were incubated with EGCG (0.1 and 5 microM) and TA (1 and 5 microM) for 30 min on three consecutive days. On the 3rd day, DNA damage was introduced with metabolically activated tobacco carcinogen benzo[a]pyren-7,8-dihydrodiol-9,10-epoxid (BPDE) [9 microM] for 60 min. The resulting DNA damage was measured with alkaline single-cell microgel electrophoresis (comet assay) and quantified using the olive tail moment (OTM). By incubating MOC with the polyphenols, the DNA damage caused by BPDE was significantly decreased at all concentrations. To our knowledge, this is the first test using cell cultures produced from fresh biopsies that demonstrates ECGC and TA as promising chemopreventive agents and confirms nutritional studies.

Cumulative genotoxic and apoptotic effects of xenobiotics in a mini organ culture model of human nasal mucosa as detected by the alkaline single cell microgel electrophoresis assay and the annexin V-affinity assay

Three-dimensional mini organ cultures of human inferior nasal turbinate epithelia have proved to be a useful tool in genotoxicology studies. They allow repetitive or chronic exposure of cells to xenobiotics in a well-preserved organ-specific mucosal architecture for an extended period of time. It is the aim of the present study to concurrently monitor cumulative genotoxic and apoptotic effects of sodium dichromate, N-nitrosodiethylamine (NDEA) and N-methyl- N-nitro- N-nitroso-guanidine (MNNG). Mini organs were raised by separating fresh specimens of human inferior nasal turbinates ( n = 11) into 1 mm 3 sized pieces and culturing them on multiwell plates with bronchial epithelial basal medium for 6 days. Aliquots of the mini organs were subsequently exposed to sodium dichromate (1.0 mM, 1 h), NDEA (50 mM, 1 h) or MNNG (0.07 mM, 1 h) on days 7, 9 and 11 versus a single exposure on day 11 only. DNA fragmentation and apoptotic events were assessed on day 11 using the alkaline single cell microgel electrophoresis assay (comet assay) and the annexin V-affinity assay. Significant DNA fragmentation could be demonstrated after a single exposure of the mini organs to sodium dichromate. Following three subsequent incubations, there was a further increase in the genetic damage observed, accompanied by an increase in the rate of apoptotic cells. In contrast, after single and triple incubation with NDEA there was neither an increase in genetic damage nor in the fraction of apoptotic cells detectable. Repetitive exposure to MNNG resulted in an accumulation of DNA damage without an observable increase in apoptosis. The results verify the need to assess apoptosis in genotoxicology research and to investigate cumulative effects of xenobiotics. Three-dimensional mini organ cultures of human upper aerodigestive tract epithelia have shown to be well-suited for improving the ability to distinguish between cumulative genotoxic and apoptotic effects.

Ellagic acid, a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro: With the comparison of N-acetylcysteine

The present work is aimed at evaluating the protective effect of ellagic acid (EA), a natural polyphenolic compound that is widely distributed in fruits and nuts against nicotine-induced toxicity in rat peripheral blood lymphocytes. The effect of EA against nicotine toxicity was compared with N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine at the doses of 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM for 1 h in culture media. Thiobarbituric acid reactive substances (TBARS), a lipid peroxidative marker and reduced glutathione (GSH), as indicative of endogenous antioxidant status were analyzed to fix the optimum dose. The lowest concentration eliciting significant damage was 1 mM nicotine and maximum damage was observed with 3 mM concentration, as evidenced by increased levels of TBARS and decreased levels of GSH. Hence, the test concentration was fixed at 3 mM nicotine. To establish most effective protective support we used five different concentrations of EA (10, 50, 100, 150 and 300 μM) against 3 mM nicotine. A dose-dependent inhibitory effect was observed with all doses of EA. Maximum protection was observed at the dose of 100 μM EA. So, 100 μM dose was used for further studies. We have tested five different concentrations of NAC—0.25, 0.5, 1, 2 and 4 mM to elucidate the optimum protective dose against nicotine toxicity. One millimolar NAC showed a significant protection against nicotine toxicity. Protective effect of EA against nicotine toxicity was elucidated by analyzing the lipid peroxidative index, viz., TBARS, hydroperoxides (HP) and endogenous antioxidant status, viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), Vitamins A, E and C. DNA damage and repair were assessed by using alkaline single-cell microgel electrophoresis (Comet assay) and micronucleus assay. There was a significant increase in the levels of lipid peroxidative index, severity in DNA damage and micronuclei number in nicotine-treated group, which was positively modulated by EA treatment. Antioxidant status was significantly depleted in nicotine-treated group, which was effectively restored by EA treatment. The protection of EA against nicotine toxicity was equally effective to that of NAC. EA and NAC treatment alone did not produce any damage to the normal lymphocytes at their effective doses. These findings suggest the potential use and benefit of EA as a modifier of nicotine-induced genotoxicity.

Trägt Nikotin zur Krebsentstehung im oberen Aerodigestivtrakt bei?

It is accepted that nicotine in tobacco smoke causes addiction via nicotinic acetylcholine receptors in the central nervous system. For a long time, the tumorigenic potential of smoking was attributed to compounds other than nicotine. However, more recently data have accumulated which suggest that nicotine may add to the cancer risk by stimulating cellular growth via non-neuronal acetylcholine receptors, by suppressing apoptosis, and by inducing angiogenesis not only in atheromatous plaques but also in tumors. In the present study the possible direct genotoxic effects of nicotine on DNA were investigated in human target cells of carcinogenesis in the upper aerodigestive tract. Human nasal mucosa, lymphatic tissue of the palatine tonsils, supraglottic epithelium of the larynx, and peripheral lymphocytes were exposed to rising concentrations of nicotine. DNA damage was investigated by alkaline single-cell microgel electrophoresis (Comet) assay. Cytotoxicity was assessed by trypan blue exclusion. Nicotine induced dose-dependent DNA damage in all cell types at low cytotoxic concentrations that allowed viabilities well above 80%. The lowest nicotine concentrations eliciting a significant increase in DNA migration were 1 mM for tonsillar cells and 0.25 mM for all other cell types. Nicotine induces genotoxic effects in human target cells of carcinogenesis in the upper aerodigestive tract at relevant concentrations. Thus, nicotine may contribute directly to tumor initiation resulting from smoking.

Effects of co-exposure to extremely low frequency (50 Hz) magnetic fields and xenobiotics determined in vitro by the alkaline comet assay

In the present study, we used human peripheral blood leukocytes from 4 different donors, to investigate in vitro the possible genotoxic and/or co-genotoxic activity of extremely low frequency magnetic fields (ELF-MF) at 3 mT intensity. Two model mutagens were used to study the possible interaction between ELF-MF and xenobiotics: N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and 4-nitroquinoline N-oxide (4NQO). Primary DNA damage was evaluated by the alkaline single-cell microgel-electrophoresis (“comet”) assay. Control cells (leukocytes not exposed to ELF-MF, nor treated with genotoxins) from the different blood donors showed a comparable level of basal DNA damage, whereas the contribution of individual susceptibility toward ELF-MF and the tested genotoxic compounds led to differences in the extent of DNA damage observed following exposure to the genotoxins, both in the presence and in the absence of an applied ELF-MF. A 3 mT ELF-MF alone was unable to cause direct primary DNA damage. In leukocytes exposed to ELF-MF and genotoxins, the extent of MNNG-induced DNA damage increased with exposure duration compared to sham-exposed cells. The opposite was observed in cells treated with 4NQO. In this case the extent of 4NQO-induced DNA damage was somewhat reduced in leukocytes exposed to ELF-MF compared to sham-exposed cells. Moreover, in cells exposed to ELF-MF an increased concentration of GSH was always observed, compared to sham-exposed cells. Since following GSH conjugation the genotoxic pattern of MNNG and 4NQO is quite different, an influence of ELF-MF on the activity of the enzyme involved in the synthesis of GSH leading to different activation/deactivation of the model mutagens used was hypothesized to explain the different trends observed in MNNG and 4NQO genotoxic activity in the presence of an applied ELF-MF. The possibility that ELF-MF might interfere with the genotoxic activity of xenobiotics has important implications, since human populations are likely to be exposed to a variety of genotoxic agents concomitantly with exposure to this type of physical agent.

The Tobacco Alkaloid Nicotine Demonstrates Genotoxicity in Human Tonsillar Tissue and Lymphocytes

Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.

Effects of co-exposure to extremely low frequency (ELF) magnetic fields and benzene or benzene metabolites determined in vitro by the alkaline comet assay

In the present study, we investigated in vitro the possible genotoxic and/or co-genotoxic activity of 50 Hz (power frequency) magnetic fields (MF) by using the alkaline single-cell microgel-electrophoresis (comet) assay. Sets of experiments were performed to evaluate the possible interaction between 50 Hz MF and the known leukemogen benzene. Three benzene hydroxylated metabolites were also evaluated: 1,2-benzenediol (1,2-BD, catechol), 1,4-benzenediol (1,4-BD, hydroquinone), and 1,2,4-benzenetriol (1,2,4-BT). MF (1 mT) were generated by a system consisting of a pair of parallel coils in a Helmholtz configuration. To evaluate the genotoxic potential of 50 Hz MF, Jürkat cell cultures were exposed to 1 mT MF or sham-exposed for 1 h. To evaluate the co-genotoxic activity of MF, the xenobiotics (benzene, catechol, hydroquinone, and 1,2,4-benzenetriol) were added to Jürkat cells subcultures at the beginning of the exposure time. In cell cultures co-exposed to 1 mT (50 Hz) MF, benzene and catechol did not show any genotoxic activity. However, co-exposure of cell cultures to 1 mT MF and hydroquinone led to the appearance of a clear genotoxic effect. Moreover, co-exposure of cell cultures to 1 mT MF and 1,2,4-benzenetriol led to a marked increase in the genotoxicity of the ultimate metabolite of benzene. The possibility that 50 Hz (power frequency) MF might interfere with the genotoxic activity of xenobiotics has important implications, since human populations are likely to be exposed to a variety of genotoxic agents concomitantly with exposure to this type of physical agent.

DNA repair capacity in lymphocytes of nasopharyngeal cancer patients

Possible hereditary factors in the tumorigenesis of nasopharyngeal cancer (NPC) have not yet been clearly identified. In the present study, the DNA repair capacity of lymphocytes after exposure to the nitrosamine NDEA was quantified in order to elucidate whether this measure may be a factor in susceptibility to NPC. The alkaline single-cell microgel electrophoresis (Comet) assay was used to quantify chemically induced DNA damage and repair capacity in lymphocytes of 30 NPC patients (NPC) and 29 non-tumor donors (NTD). The induction of DNA single strand breaks, alkali labile and incomplete excision repair sites after exposure of lymphocytes to NDEA was assessed as differences between repair intervals of 0 min, 15 min, 30 min and 60 min, respectively. A RC(total) was assessed using the difference between the OTMs of 0 min of repair time and the 60-min repair interval for both groups. Repair capacities (RC) were calculated for the intervals according to the Olive Tail Moment (OTM), a quantitative measure for DNA migration in the Comet assay for the group of NPC patients and the NTD, accordingly. RCs were compared between the two groups using the Mann-Whitney U-Test. RC(15 min), RC(30 min) RC(60 min) and the RC(total) after a 60-min repair interval demonstrated no significant difference between the two groups. Furthermore, when comparing grades of DNA migration (OTM<2, 2-5, 5-10, 10-20, 20-30 and >30), there were no differences evident. In this investigation, rejoining of DNA single strand breaks in lymphocytes of NPC and NTD appeared to be accomplished to an equal degree and in equal time periods. However, the applied method does not give evidence concerning the quality of the single strand break rejoining processes. In this group of patients, tumorigenesis in NPC could not be associated with a decreased DNA repair capacity.

Genotoxic effects of myosmine in human lymphocytes and upper aerodigestive tract epithelial cells.

Myosmine, 3-(1-pyrroline-2-yl)pyridine, is an alkaloid found in tobacco plants. Recently, it was also detected in various edibles and staple foods. Whereas other tobacco alkaloids such as nicotine and nornicotine and their nitrosation products, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), have been widely discussed, the mutagenic impact of myosmine has not been investigated in detail. In the present study, possible genotoxic effects of myosmine were studied in human lymphocytes and nasal mucosal cells using the alkaline single cell microgel electrophoresis (Comet) assay. DNA single strand breaks, alkali labile sites and incomplete excision repair sites were expressed using the Olive tail moment (OTM). One hour incubation with myosmine at 0, 5, 10, 25 and 50 mM induced a low but significantly dose-dependent increase of DNA migration from 1.29 +/- 0.13 to 18.25 +/- 1.59 (OTM, mean +/- S.E., N=11) in lymphocytes. In nasal mucosal cells a similar although somewhat less extensive DNA damage from 1.17 +/- 0.12 to 21.67 +/- 2.97 (OTM, mean +/- S.E., N=10-11) was obtained after 1 h incubation with myosmine at 0, 10, 25, 50 and 100 mM. After prolonged incubation of human lymphocytes with 10mM myosmine for 1, 3, 6, and 24 h, a significant time-dependent increase of DNA migration from 3.45 +/- 0.43 to 57.77 +/- 8.24 (OTM, mean +/- S.E., N=4) was observed. Our data indicate that myosmine expresses significant genotoxic effects in human target cells of carcinogenesis. This result warrants further investigations on the impact of this dietary component on human health.

Pesticide-induced primary DNA damage in peripheral blood leukocytes of farm workers evaluated by the computerized ‘comet’ assay

The alkaline single-cell microgel-electrophoresis (comet') assay, allowing the evaluation of damage occurring at the DNA level in individual cells associated with exposure to genotoxic compounds, appears to be a promising tool for human biomonitoring studies. In a molecular epidemiology approach aimed at evaluating the eventual genetic damage induced by occupational exposure to pesticides, peripheral blood leukocytes of 17 male farm workers and of 17 age-matched controls living in the same area were analysed for the presence of DNA damage using the 'comet' assay. In this paper we report results obtained after a re-analysis by a computerized system of photomicrographs previously used for the direct manual measurement of comet length. Exposed subjects (farm workers) showed a statistically significant increase in the number of damaged cells (cells with an abnormal size tail, AST) for the tail length parameter, as compared with controls. We did not observe any statistically significant effect of cigarette smoking on the extent of DNA damage (both exposed and controls) and trends similar to those obtained for the groups in toto were observed when smoking habits was considered. Our results support data reported in the literature indicating that occupational exposure to pesticides may evoke some genotoxic effect and, consequently, confirm the necessity to improve preventive measures and to perform accurate health surveillance of individuals exposed to pesticides.

In vitro genotoxicity of terbutryn evaluated by the alkaline single-cell microgel-electrophoresis "comet" assay.

Terbutryn, a s-triazine herbicide, is extensively used in agriculture as a selective pre- and postemergence control agent for most grasses and many annual broadleaf weeds in cereal and legume fields, and under fruit trees. Terbutryn was reported to degrade slowly, with half-lives of 240 and 180 days in pond and river sediment, respectively. The tendency of this herbicide to move from treated soils to water compartments through water runoff and leaching was demonstrated and residual amounts of terbutryn and its metabolites have been found in drinking water, and industrial food products, long after application. Although this herbicide may be regarded as a contaminant of our environment, only limited and inconsistent data exist concerning its genotoxic properties. In this study, the DNA-damaging ability of the herbicide was evaluated in the alkaline single-cell microgel-electrophoresis ("comet") assay by testing terbutryn in the presence of S9mix (rat liver homogenate containing microsomal enzymes plus cofactors) prepared with liver homogenate from both uninduced (basal) and aroclor 1254-induced rats. DNA damage was recorded in freshly isolated human peripheral blood leukocytes. A statistically significant increase in the extent of primary DNA damage, more pronounced in the absence of S9mix, took place only when terbutryn concentrations were high (100 and 150 microg/ml), in the presence of a concomitant mild cytotoxic effect.

On the mechanism of cogenotoxic action between ingested amphibole asbestos fibres and benzo[a]pyrene: II. Tissue specificity studies using comet assay

Epidemiological data seem to be equivocal on the probable increase in cancer incidence in populations exposed to asbestos-fibre contaminated drinking water. Although animal experiments failed to demonstrate carcinogenicity of oral asbestos exposure, the large surface area of the fibres, however, creates the possibility of cogenotoxic action with adsorbed water-borne organics. In our animal model, rats were gavaged with untreated UICC crocidolite and anthophyllite fibres and fibres that had been allowed to adsorb benzo[a]pyrene molecules from aqueous solutions. Peritoneal macrophages and intestine, parietal peritoneum and omentum samples were obtained from the animals after 24 h. The alkaline single-cell microgel electrophoresis assay (comet assay) was performed on cells isolated from the solid tissues. Tail moment was applied as a basis of evaluation following image analysis. Our results indicate high levels of DNA strand breaks in the cells prepared from the omentum and intestine. We could also demonstrate a significant potentiating effect of the adsorbed carcinogen on the induction of DNA damage in the omentum. The parietal peritoneum and macrophages were not involved in the early genotoxic alterations under study. Our results support the molecular model of asbestos cocarcinogenesis, including both asbestos-induced deletions and mutations caused by a mutagen carried by the same fibres.