Genotoxic evaluation of the antimicrobial drug, trimethoprim, in cultured human lymphocytes

The antimicrobial drug, trimethoprim, was evaluated for genotoxicity in human peripheral blood lymphocyte cultures set-up from two healthy donors. Sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. The treatment was done using different trimethoprim concentrations ranging from 1 to 100 microg/ml. From our results, we can conclude that this drug is able to induce both cytotoxic and moderate genotoxic effects, as revealed by the increases seen in SCE and MN frequencies in cultures from the two donors and, at least, at one of the concentrations tested.     

Genotoxic evaluation of the antimicrobial drug, trimethoprim, in cultured human lymphocytes

The antimicrobial drug, trimethoprim, was evaluated for genotoxicity in human peripheral blood lymphocyte cultures set-up from two healthy donors. Sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. The treatment was done using different trimethoprim concentrations ranging from 1 to 100 μg/ml. From our results, we can conclude that this drug is able to induce both cytotoxic and moderate genotoxic effects, as revealed by the increases seen in SCE and MN frequencies in cultures from the two donors and, at least, at one of the concentrations tested.     

Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes

The genotoxicity of the antimicrobial drug sulfamethoxazole was evaluated in cultured human peripheral blood lymphocytes. The frequencies of sister-chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests cover a wide range of induced genetic damage such as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up with blood samples from two healthy donors and the treatment was done with different sulfamethoxazole concentrations ranging from 10 to 500 microg/ml. From the results obtained it appears that this drug is able to induce weak genotoxic effects, as revealed by the slight increase in the SCE and MN frequencies, at least at one of the two highest concentrations tested. However, the results of the SCE assay should be interpreted with caution because the increase is just significant. In addition, cyotoxic/cytostatic effects of sulfamethoxazole were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI).     

Lack of genotoxic effects (micronucleus induction) in human lymphocytes exposed in vitro to 900 MHz electromagnetic fields

In the present study, we investigated the induction of genotoxic effects in human peripheral blood lymphocytes after exposure to electromagnetic fields used in mobile communication systems (frequency 900 MHz). For this purpose, the incidence of micronuclei was evaluated by applying the cytokinesis-block micronucleus assay. Cytotoxicity was also investigated using the cytokinesis-block proliferation index. The experiments were performed on peripheral blood from 20 healthy donors, and several conditions were tested by varying the duration of exposure, the specific absorption rate (SAR), and the signal [continuous-wave (CW) or GSM (Global System of Mobile Communication) modulated signal]. The following exposures were carried out: (1) CW intermittent exposure (SAR = 1.6 W/kg) for 6 min followed by a 3-h pause (14 on/off cycles); (2) GSM signal, intermittent exposure as described in (1); (3) GSM signal, intermittent exposure as described in (1) 24 h before stimulation with phytohemagglutinin (8 on/off cycles); (4) GSM signal, intermittent exposure (SAR = 0.2 W/kg) 1 h per day for 3 days. The SARs were estimated numerically. No statistically significant differences were detected in any case in terms of either micronucleus frequency or cell cycle kinetics.     

Inhalation of formaldehyde does not induce genotoxic effects in broncho-alveolar lavage (BAL) cells of rats

Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential genotoxic effects in the lung were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. Broncho-alveolar lavage (BAL) cells were obtained by lung lavage with physiological saline and counted. From one half of the cells, slides for the micronucleus test (MNT) were prepared by cytocentrifugation; with the other half, the comet assay was performed. DNA migration in the comet assay was measured both directly and after irradiation of the cells with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA-protein cross-links (DPX). For the comet assay, four slides were analysed from each cell sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis and tail intensity (% tail DNA) and tail moment were evaluated. The frequency of micronucleated BAL cells was determined in acridine orange-stained slides by analysing 2000 cells per animal. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28 days study with FA concentrations up to 15 ppm does not lead to genotoxic effects in BAL cells of rats. Because detection of DPX by the comet assay is a very sensitive biomarker of FA exposure of cells, our results suggest that there is no genetically relevant exposure of the lung after FA inhalation. The results of our inhalation study, which was performed under GLP conditions, call into question the biological significance of previously reported genotoxic effects in the lung of rats after FA inhalation.     

Interlaboratory reproducibility of atypical glandular cells of undetermined significance: a national survey

OBJECTIVE: The aim of this study was to evaluate the inter-laboratory reproducibility for atypical glandular cells (AGC) (The Bethesda System (TBS) 2001) of the laboratories involved in the screening programmes in Italy. METHODS: A set of 35 selected slides were circulated among 167 laboratories involved in local population-based cervical screening programmes. Each laboratory provided one single diagnosis per smear. The smears were read blind to the original diagnosis and to the diagnoses provided by other laboratories. A 'majority' diagnosis was defined for each case and assumed as the reference standard. The diagnosis provided from each laboratory was compared with the majority diagnosis. RESULTS: According to the majority report the 35 slides in the set were classified as negative in nine cases, AGC in eight, adenocarcinoma in eight, and squamous lesion or squamous + glandular lesion in 10. The crude agreement between all pairs of laboratories was 49.43%. K-values were 0.46, 0.21, 0.34, 0.36 and 0.32 for negative, AGC/AIS (adenocarcinoma in situ of endocervix), AdenoCa, Sq/Sq + Gl and all reporting categories respectively. Concordance according to overall K was moderate to substantial in 77% of the participating laboratories. CONCLUSIONS: The present study shows that the AGC category is not easily reproducible. The data confirmed the importance, in a screening scenario, of AGC/AIS diagnoses, but also presented difficulties in differentiating between the two diagnoses. In addition to the results obtained from the circulation of the slides, laboratories which had annually a low number of cervical smears were able to gain experience focused on particular morphological pictures.     

Genetic damage in exfoliated cells from oral mucosa of individuals exposed to X-rays during panoramic dental radiographies

The genotoxic effects of X-ray emitted during dental panoramic radiography were evaluated in exfoliated cells from oral epithelium through a differentiated protocol of the micronucleus test. Thirty-one healthy individuals agreed to participate in this study and were submitted to this procedure for diagnosis purpose after being requested by the dentist. All of them answered a questionnaire before the examination. Cells were obtained from both sides of the cheek by gentle scrapping with a cervical brush, immediately before the exposure and after 10 days. Cytological preparations were stained according to Feulgen-Rossenbeck reaction and analyzed under light and laser scanning confocal microscopies. Micronuclei, nuclear projections (buds and broken eggs) and degenerative nuclear alterations (condensed chromatin, karyolysis and karyorrhexis) were scored. The frequencies of micronuclei, karyolysis and pycnosis were similar before and after exposure (P > 0.90), whereas the condensation of the chromatin and the karyorrhexis increased significantly after exposure (P < 0.0001). In contrast, both bud and broken egg frequencies were significantly higher before the examination (P < 0.005), suggesting that these structures are associated to the normal epithelium differentiation. The results suggest that the X-ray exposure during panoramic dental radiography induces a cytotoxic effect by increasing apoptosis. We also believe that the score of other nuclear alterations in addition to the micronucleus improves the sensitivity of genotoxic effects detection.     

Study of p53 expression and post‐transcriptional modifications after GSM‐900 radiofrequency exposure of human amniotic cells

The potential effects of radiofrequency (RF) exposure on the genetic material of cells are very important to determine since genome instability of somatic cells may be linked to cancer development. In response to genetic damage, the p53 protein is activated and can induce cell cycle arrest allowing more time for DNA repair or elimination of damaged cells through apoptosis. The objective of this study was to investigate whether the exposure to RF electromagnetic fields, similar to those emitted by mobile phones of the second generation standard, Global System for Mobile Communications (GSM), may induce expression of the p53 protein and its activation by post‐translational modifications in cultured human cells. The potential induction of p53 expression and activation by GSM‐900 was investigated after in vitro exposure of human amniotic cells for 24 h to average specific absorption rates (SARs) of 0.25, 1, 2, and 4 W/kg in the temperature range of 36.3–39.7 °C. The exposures were carried out using a wire‐patch cell (WPC) under strictly controlled conditions of temperature. Expression and activation of p53 by phosphorylation at serine 15 and 37 were studied using Western blot assay immediately after three independent exposures of cell cultures provided from three different donors. Bleomycin‐exposed cells were used as a positive control. According to our results, no significant changes in the expression and activation of the p53 protein by phosphorylation at serine 15 and 37 were found following exposure to GSM‐900 for 24 h at average SARs up to 4 W/kg in human embryonic cells. Bioelectromagnetics 34:52–60, 2013. © 2012 Wiley Periodicals, Inc.     

Specific absorption rate in rats exposed to 2,450-MHz microwaves under seven exposure conditions

Both positive and negative biological effects of microwaves on drug actions in rats exposed to 1-mW/cm2, 2,450-MHz microwaves have been reported by several investigators. We conducted dosimetry studies for seven different exposure conditions to determine whether these different results could be due to the rats having been exposed differently. They included anterior and posterior exposures in a circular waveguide, near field, far field with E- or H-field parallel to the long axis of the body and dorsal exposure in a miniature anechoic chamber with E- or H-field parallel to the long axis of the body. The average specific absorption rates (SARs) in the head, tail, and body of the exposed rats were measured by means of a calorimetry system. The local SARs at eight locations in the brain were determined by temperature measurement with Vitek probes. Intensive coupling of energy to the tail when it was exposed parallel to the E-field was shown by thermography. For the same average incident power density, the average SARs in the heads of rats were about two times higher in the circular waveguide than for other exposures. The local SARs in the brain varied for different exposure conditions. Statistical comparisons of SARs under the different exposure conditions are presented.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

Frequency of chromosomal aberrations in a subject accidentally exposed to 137Cs in the Goiania (Brazil) radiation accident: intercomparison among four laboratories

After the Goiania radiation accident which occurred in Brazil in September 1987, an intercomparison was performed to determine whether different cytogenetic laboratories would score similar frequencies of chromosomal aberrations in cultures of lymphocytes of a highly exposed patient. For this purpose 2 chromosome slides from the subject were scored by 4 laboratories in total. The results were consistently close and confirmed the high frequency of chromosome-type aberrations observed initially in the patient.     

Tradescantia stamen hair mutation bioassay

The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100 μg/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45 μg/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80 μg/ml. NaN₃, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN₃ ranged between 3 and 80 μg/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well.     

Individual responsiveness to induction of micronuclei in human lymphocytes after exposure in vitro to 1800-MHz microwave radiation

The widespread application of microwaves is of great concern in view of possible consequences for human health. Many in vitro studies have been carried out to detect possible effects on DNA and chromatin structure following exposure to microwave radiation. The aim of this study is to assess the capability of microwaves, at different power densities and exposure times, to induce genotoxic effects as evaluated by the in vitro micronucleus (MN) assay on peripheral blood lymphocytes from nine different healthy donors, and to investigate also the possible inter-individual response variability. Whole blood samples were exposed for 60, 120 and 180 min to continuous microwave radiation with a frequency of 1800 MHz and power densities of 5, 10 and 20 mW/cm(2). Reproducibility was tested by repeating the experiment 3 months later. Multivariate analysis showed that lymphocyte proliferation indices were significantly different among donors (p<0.004) and between experiments (p<0.01), whereas the applied power density and the exposure time did not have any effect on them. Both spontaneous and induced MN frequencies varied in a highly significant way among donors (p<0.009) and between experiments (p<0.002), and a statistically significant increase of MN, although rather low, was observed dependent on exposure time (p=0.0004) and applied power density (p=0.0166). A considerable decrease in spontaneous and induced MN frequencies was measured in the second experiment. The results show that microwaves are able to induce MN in short-time exposures to medium power density fields. Our data analysis highlights a wide inter-individual variability in the response, which was confirmed to be a characteristic reproducible trait by means of the second experiment.     

Genotoxic Effects of Amplitude-Modulated Microwaves on Human Lymphocytes Exposed in Vitro under Controlled Conditions

Peripheral human blood from 23 healthy donors aged between 23 and 95 years was exposed to continuous wave (CW) or 50 Hz amplitude modulated (AM) microwave radiation and was cultured for 72 h. Other exposure parameters were: frequency 9 GHz, specific absorption rate (SAR) 90 mW/g, exposure duration 10 min. The possible genotoxic effect was evaluated by means of cytokinesis-block micronucleus method. A significant (p < 0.05) increase in micronuclei was found following AM microwave exposure.     

Microarray gene expression profiling of a human glioblastoma cell line exposed in vitro to a 1.9 GHz pulse-modulated radiofrequency field

The widespread use of mobile phones has led to public concerns about the health effects associated with exposure to radiofrequency (RF) fields. The paramount concern of most persons relates to the potential of these fields to cause cancer. Unlike ionizing radiation, RF fields used for mobile telecommunications (800-1900 MHz) do not possess sufficient energy to directly damage DNA. Most rodent bioassay and in vitro genotoxicity/mutation studies have reported that RF fields at non-thermal levels have no direct mutagenic, genotoxic or carcinogenic effects. However, some evidence has suggested that RF fields may cause detectable postexposure changes in gene expression. Therefore, the purpose of this study was to assess the ability of exposure to a 1.9 GHz pulse-modulated RF field for 4 h at specific absorption rates (SARs) of 0.1, 1.0 and 10.0 W/kg to affect global gene expression in U87MG glioblastoma cells. We found no evidence that non-thermal RF fields can affect gene expression in cultured U87MG cells relative to the nonirradiated control groups, whereas exposure to heat shock at 43 degrees C for 1 h up-regulated a number of typical stress-responsive genes in the positive control group. Future studies will assess the effect of RF fields on other cell lines and on gene expression in the mouse brain after in vivo exposure.     

Immunocytochemical staining of effusions; an external quality control study in The Netherlands

Immunocytochemical staining of effusions; an external quality control study in The Netherlands
In The Netherlands an external quality control study of immunocytochemical (IC) staining of effusions was initiated, consisting of three test rounds. The 12 participating laboratories received samples of malignant effusions (runs 1, 2 and 3), and five unstained control specimens prepared from the same material in runs 2 and 3. The laboratories used their own protocols to prepare and stain the samples ('in‐house' specimens). Two persons viewed and scored the slides following preset criteria concerning number and morphology of diagnostic cells, background staining and staining specificity. Better scoring results were found for control specimens, compared with 'in‐house' specimens, primarily caused by cell loss in the latter. This finding underlines the view that high quality IC needs well organized processing and staining procedures, and warrants external quality control systems.     

P3-P4 ureas and reverse carbamates as potent HCV NS3 protease inhibitors: Effective transposition of the P4 hydrogen bond donor

A series of tripeptidic acylsulfonamide inhibitors of HCV NS3 protease were prepared that explored structure-activity relationships (SARs) at the P4 position, and their in vitro and in vivo properties were evaluated. Enhanced potency was observed in a series of P4 ureas; however, the PK profiles of these analogues were less than optimal. In an effort to overcome the PK shortcomings, modifications to the P3-P4 junction were made. This included a strategy in which one of the two urea N–H groups was either N-methylated or replaced with an oxygen atom. The former approach provided a series of regioisomeric N-methylated ureas while the latter gave rise to P4 reverse carbamates, both of which retained potent NS3 inhibitory properties while relying upon an alternative H-bond donor topology. Details of the SARs and PK profiles of these analogues are provided.     

Evaluation of genotoxic effect of low level 50 Hz magnetic fields on human blood cells using different cytogenetic assays

The question whether extremely low frequency magnetic fields (ELFMFs) may contribute to mutagenesis or carcinogenesis is of current interest. In order to evaluate the possible genotoxic effects of ELFMFs, human blood cells from four donors were exposed in vitro for 48 h to 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system. Comet assay (SCGE), sister chromatid exchanges (SCE), chromosome aberrations (CAs), and micronucleus (MN) test were used to assess the DNA damage. ELF pretreated cells were also irradiated with 1 Gy of X-ray to investigate the possible combined effect of ELFMFs and ionizing radiation. Furthermore, nuclear division index (NDI) and proliferation index (PRI) were evaluated. Results do not evidence any DNA damage induced by ELFMF exposure or any effect on cell proliferation. Data obtained from the combined exposure to ELFMFs and ionizing radiation do not suggest any synergistic or antagonistic effect.     

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.