DNA damage in mononuclear leukocytes of farmers measured using the alkaline comet assay: discussion of critical parameters and evaluation of seasonal variations in relation to pesticide exposure.

The alkaline comet assay was used to quantify, using visual and image analyses, the level of DNA damage in mononuclear leukocytes of farmers who were occupationally exposed to pesticides. Hematological parameters were also measured on the same samples. Enrollment of farmers was based on handling of heavily used pesticides at particular periods during one spraying season. Forty-one blood samples from 29 different farmers were collected at the beginning of the season (n = 11) and at the intermediate (n = 14) and final (n = 16) periods of intense spraying activity. The mean numbers of lymphocytes and eosinophils were nonsignificantly higher in groups 3, 1, and 4 than they were in group 2. No individual characteristics significantly influenced the mean number of lymphocytes or eosinophils, and no correlation was observed between pesticide exposure-related parameters and hematological parameters. The level of DNA damage was significantly (P < 0.01) higher in groups 3, 1, and 4 than it was in group 2. In addition, DNA damage quantification was not significantly different among investigators or among slides. Prescription medicine, alcohol consumption, and age had no statistically significant effect on DNA damage level. Conversely, smoking (smokers versus non- and ex-smokers) significantly influenced DNA damage level (P < 0.0001). A significant (P < 0.05) negative correlation was detected between the number of days without pesticide spraying and DNA damage level, particularly among non- and ex-smokers. DNA damage detected by the alkaline comet assay seems to reflect ongoing exposure to genotoxic agents but not an accumulation of damage.

DNA damage in mononuclear leukocytes of farmers measured using the alkaline comet assay: modifications of DNA damage levels after a one-day field spraying period with selected pesticides.

The alkaline comet assay was used to assess DNA damage in mononuclear leukocytes of farmers before and after a 1-day spraying period with selected pesticides under usual conditions. Two blood samples were collected, one in the morning of the day of spraying (S0) and the second in the morning of the day after (S1). Here, we assessed variations in DNA damage levels between these two sampling times. Four groups of farmers were formed, according to exposure to: (a) various fungicide-insecticide mixtures (including chlorothalonil; group 1, n = 8), (b) the herbicide isoproturon (group 2, n = 11), (c) fungicide triazoles (group 3, n = 14), and (d) a fungicide (chlorothalonil)-insecticide mixture (group 4, n = 8). An increase in DNA damage levels was observed at S1 for groups 1 and 4, who were exposed to similar pesticides. This increase was correlated with area sprayed between S0 and S1 and with the number of spraying tanks used over this 1-day period. No effect was observed on cell viability or on hematological parameters for these two groups. No statistically significant modification of DNA damage level was observed the day after spraying for groups 2 and 3, when each was observed as a whole. However, some farmers presented significantly more DNA damage after exposure, and others presented less damage. In these two groups, a significant decrease of neutrophils was observed at S1, and a decrease of red blood cells was observed in group 3. In parallel, a significant loss of lymphocyte viability was observed in these two groups. A 1-day spraying period seems to be sufficient to significantly modify DNA damage levels in mononuclear leukocytes, but the correlation of this change with pesticide-related exposure parameters depends on the kind of pesticide concerned.

Effects of melatonin on N-nitroso-N-methylurea-induced carcinogenesis in rats and mutagenesis in vitro (Ames test and COMET assay).

The effect of melatonin, an indole hormone of the pineal gland, on the initiation of N-nitroso-N-methylurea (NMU)-induced carcinogenesis in rats and mutagenesis in vitro has been investigated. Two-month-old female LIO rats (groups 1 and 2) were exposed to a single injection of NMU (50 mg/kg of body weight, i.v.). Rats from group 2 were given melatonin orally (20 mg/l) from 18:00 to 09:00 h over 3 days (2 days before and 1 day after NMU injection). Animals from group 1 (control) were administered the solvent (ethanol/water, 1:1000). Rats were followed up to natural death or were sacrificed when moribund. Tumors developed both in rats treated with NMU alone (50.0%) and in rats exposed to NMU plus melatonin (34.8%). The percentage of malignant tumor-bearing rats in group 2 (21.7%) was lower (P < 0.02) than that in the other group (41.7%). Melatonin also decreased the multiplicity of malignant tumors 1.3-fold and reduced the incidence of malignancies in some organs. Two in vitro tests were used for mutagenesis studies: the Ames test (strains TA 100 and TA 102 of Salmonella typhimurium) and the Single Cell Gel Electrophoresis assay (SCGE assay or COMET assay) performed on CHOK1 cells. Melatonin itself revealed no genotoxic effect in either of the tests. No protective action of melatonin (at doses of up to 2 micromol/plate) towards NMU was found in the Ames test. In contrast, in the SCGE assay a slight, but statistically significant (P < 0.001), dose-related anticlastogenic effect of melatonin (10(-10)-10(-7) M) was observed. Thus, our data indicate that melatonin may act as an anti-initiating hormone in NMU-induced carcinogenesis and possess anticlastogenic activity towards NMU in CHOK1 cells.

Inhibitory effect of melatonin on 7, 12-dimethylbenz[a]anthracene-induced carcinogenesis of the uterine cervix and vagina in mice and mutagenesis in vitro.

Forty female CBA mice aged 3-4 months were exposed twice a week during 2 months to intravaginal applications of polyurethane sponges impregnated with 0.1% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in triethyleneglycol. Three hours after each application the sponges were taken out. Starting from the day of the 1st DMBA application a part of mice was exposed five times a week during 4 months with melatonin in tap water (20 mg/l) given at night time (from 18:00 to 09:00 h). Additional 20 female CBA mice were intact and served as a control. All mice were sacrificed in 6 months after start of the experiment. Seven of 20 mice exposed to DMBA alone developed malignancies in the vagina and cervix uteri and two mice developed benign cervical tumors. No malignancies in vagina and uterine cervix and three vaginal papillomas were observed in mice exposed to DMBA+melatonin. There were no any tumors in intact controls. Two in vitro tests were used for mutagenicity studies: the Ames test (strains TA 97 and TA 98 of Salmonella typhimurium) and the single cell gel electrophoresis assay (SCGE assay or COMET assay) performed on CHOK1 cells. In tested strains melatonin significantly reduced the mutagenicity of DMBA. In the SCGE assay preincubation with melatonin led to a strong inhibition of clastogenic activities of DMBA. Thus, our data indicate that pineal indole hormone melatonin inhibits cervical and vaginal carcinogenesis induced by DMBA in mice and possess antimutagenic and anticlastogenic effect in vitro.

Modulatory effects of melatonin on genotoxic response of reference mutagens in the Ames test and the comet assay.

The effect of a potent endogenous antioxidant, the pineal gland indole melatonin (MLT) on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay (SCGE assay or COMET assay). The 12 mutagens used were 7, 12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene (AF), 1,2-dimethylhydrazine (DMH), bleomycin, cyclophosphamide (CP), 4-nitroquinoline-N-oxide (NQO), 2,4, 7-trinitro-9-fluorenone (TNF), 9-aminoacridine (AA), N-nitrosomethylurea (NMU), mitomycin C and sodium azide tested in the absence or in the presence of S9 mix. MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 microM) in the SCGE assay. In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation. In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU. With mitomycin C, MLT exacerbated responses in both tests. The possible mechanisms of MLT's inhibitory action are discussed.

Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorothalonil) in human lymphocytes with the comet assay.

The effects of two fungicides (carbendazim and chlorothalonil) on the induction of DNA damage in human peripheral blood lymphocytes (human PBL) have been investigated using the single cell gel electrophoresis assay (SCGE assay or comet assay) immediately after a 1-h treatment and after a 24-h post-treatment incubation. The assessment of etoposide (an effective antitumour agent) effects on human PBL in terms of cell viability and dose-DNA damage relationships was made and etoposide selected as a positive control. The results indicate that etoposide induces significant (p < 0.01) dose-dependent DNA damages for concentrations at which the loss of cell viability is low. After a 24-h recuperation period, all observed DNA damages has disappeared. With SCGE assay performed after a 1-h treatment, similar positive results were observed with chlorothalonil alone or in association with carbendazim, without any loss of cell viability. However, a dramatic loss of cell viability was measured after 24 h and was associated with a large proportion of highly damaged cells. In contrast, carbendazim was not cytotoxic on human PBL and did not induced DNA damage using the SCGE assay either immediately after treatment or after a 24-h post-treatment incubation. These results point to the necessity of an adequate evaluation of immediate and long-term cytotoxicity of compounds that are to be assessed by the SCGE assay.

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells.

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.

Lack of genotoxicity of potassium iodate in the alkaline comet assay and in the cytokinesis-block micronucleus test. Comparison to potassium bromate.

Iodine could be added to the diet of human population in the form of iodide or iodate but iodate had not been adequately tested for genotoxicity and carcinogenicity. In the present study, genotoxic effects of potassium iodate were evaluated in vitro using the alkaline comet assay and the cytokinesis-block micronucleus assay on CHO cells and compared to halogenate salt analogues potassium bromate and chlorate and also to their respective reduced forms (potassium iodide, bromide and chloride). The results showed that the comet assay failed to detect the presence of DNA damage after a treatment of cells by potassium iodate for concentrations up to 10 mM. This absence of primary DNA damage was confirmed in the cytokinesis-block micronucleus assay. In the same way, results showed that potassium chlorate as well as potassium iodide, bromide and chloride did not induced DNA damage in the alkaline comet assay for doses up to 10 mM. By contrast, potassium bromate exposure led to an increase in both DNA damage and frequency of micronucleated cells. The repair of bromate-induced DNA damage was incomplete 24 h after the end of treatment. These results seem to indicate that potassium bromate would induce DNA damage by several mechanisms besides oxidative stress.

Epidemiological study of Staphylococcus aureus resistant to methicillin responsible for nosocomial infections in nine French hospitals.

A number of European studies found that nosocomial infections were caused by a limited number of methicillin-resistant Staphylococcus aureus (MRSA) strains. A study was undertaken to determine the number of MRSA clones responsible for nosocomial infections in France. Strains responsible for nosocomial infections meeting CDC criteria were collected one week every month from June to October 1997 in nine French hospitals. Strains that were positive by the oxacillin-resistance screening test were studied for the IS 431, femA, and mecA genes. Strain type was identified using pulsed-field gel electrophoresis of fragments produced by Smal digestion. Susceptibility to antimicrobials was evaluated based on inhibition zone diameters and minimal inhibitory concentrations determined using the agar dilution method. The 83 strains studied were distributed across four pulsotypes. Eleven resistance phenotypes were identified by ascending hierarchical classification based on inhibition zone diameters. MRSAs responsible for nosocomial infections belong to a limited number of clones that express variable levels of resistance to antimicrobials.

Detection by the comet assay of apoptosis induced in lymphoid cell lines after growth factor deprivation.

Dysregulation of apoptosis contributes to various diseases such as neurodegenerative or aging disorders, autoimmune syndromes or cancers. Numerous experimental paradigms have been explored to characterize molecular and cellular modulators of apoptosis. Similarly, numerous techniques have been described for detecting and/or quantifying accurately cells committed to apoptosis. Besides the conventional techniques, we describe in this report that the comet assay, which detects DNA single- and double-strand breaks in situ, at the cellular level, is relevant for the characterization of apoptotic cells. The comet assay is very sensitive and detects DNA fragmentation occurring in the apoptotic process as early as exposure of phosphatidylserine residues on the outer leaflet. Thus the comet assay can be used for the recognition of apoptosis that follows the death signal caused, for example, by genotoxic stress as well as lack of survival signal as in growth factor deprivation.

Mutagenicity of malachite green and leucomalachite green in in vitro tests.

The genotoxic potential of the fungicide malachite green (MG) and its reduced derivative leucomalachite green (LMG) was assessed in bacteria and mammalian cells using the standard Salmonella typhimurium/Ames and CHO/HGPRT tests. In vitro potential DNA damaging effects of MG and LMG were tested using the single-cell gel electrophoresis (Comet) assay on CHO cells. Malachite green was found to be extremely cytotoxic to bacteria and mammalian cells. It did not have any mutagenic activity, in any bacterial strains, in the presence or absence of metabolic activation for doses up to 10 microg per plate. In the CHO/HGPRT test, the mutagenic potential of MG could be evaluated only for very low concentrations ranging from 0.001 to 0.05 microg ml(-1) medium. When S9 fraction was added to the medium, the highest tested dose could be increased to 1 microg ml(-1). In these experimental conditions, MG did not increase the number of thioguanine-resistant mutants. Leucomalachite green was less toxic than MG to Salmonella typhimurium and did not have mutagenic activity in the Ames' test for doses up to 2000 microg per plate. It was also less cytotoxic than MG to CHO cells and was tested at doses ranging from 5 to 100 microg ml(-1). Overall results indicated that LMG was not mutagenic in the HGPRT test. In the Comet assay, MG induced DNA damage only at cytotoxic doses. Loss of cell viability was observed for doses of > or = 3 microg ml(-1), with parallel increase in DNA alterations as measured by the tail moment. After metabolic activation, however, DNA damage was observed at doses (15-20 microg ml(-1)) inducing only low cytotoxicity. In this case, the direct genotoxicity of MG metabolites could not be excluded. In the absence or presence of metabolic activation, LMG did not have any effect on cell viability or DNA damage for doses up to 500 microg ml(-1). This study indicates that LMG, which is the main residue found in fish tissues after treatment with MG, did not have any mutagenic or clastogenic effects in the experimental conditions used.

[Influence of mechanisms of Pseudomonas aeruginosa resistance on the bactericidal activity of third generation cephalosporins]. / Influence des mécanismes de résistance de Pseudomonas aeruginosa sur la bactéricidie par les céphalosporines de troisième génération.

Survey of resistance of beta-lactam's resistance of P. aeruginosa have to be conducted. 410 strains of P. aeruginosa ticarcilline resistant have been studied. 52% of these strains were cephalosporinase producing alone or in combination with penicillinase. beta-lactam's inhibitors are not fully efficient upon these strains. Upon cephalosporinase producing strains bactericidal activity of C3G is difficult to obtain. At low concentration selection of strains with upper MICs is observed. This phenomenon is not observed at high concentration of antibiotics. Cefepime show a good bactericidal activity against P. aeruginosa.

Early detection of staurosporine-induced apoptosis by comet and annexin V assays.

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.

Biological properties of 5,11-dimethyl-6H-pyrido[3,2-b]carbazoles: a new class of potent antitumour drugs.

Thirteen 5,11-dimethyl-6H-pyrido[3,2-b]carbazoles, structurally related to the antitumour drug ellipticine, were tested for their cytotoxicity against the L1210 murine leukaemia cell line and their antitumour activity against both leukaemias and solid tumours. Most of them showed an interesting antitumour activity against L1210 leukaemia, 4-hydroxy-9-chloro-2,3, 5,11-tetramethyl-6H-pyrido[3,2-b]carbazole displaying a high antitumour activity against L1210 and P388 leukaemias, B16 melanoma and M5076 sarcoma. Despite promising cytotoxic activity, 4-ethoxy-5,11-dimethyl-6H-pyrido-[3,2-b]carbazole had no antitumour activity. The ability of four drugs to induce strand breaks in DNA was studied using the single cell gel electrophoresis assay (comet assay). Most of the molecules induced DNA breaks that were totally or partially repaired after 1 h. The effects of these compounds on the L1210 cell cycle were tested as well as their abilities to induce apoptosis in these cells. Three of them induced a G2/M blockade, without any obvious evidence of apoptosis. The other compound, 4-ethoxy-5,11-dimethyl-6H-pyrido[3,2b]carbazole, did not lead to phase-specific blockade, but was a strong inductor of apoptosis in L1210 cells.