Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.

The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the determination of DNA excision repair activity in response to BPDE in different cell types.

The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.

Hyperthermia-induced genotoxic effects in human A549 cells.

Genotoxic effects of hyperthermia in vitro and in vivo have repeatedly been reported. Short-duration heat shocks and elevated temperature over longer time periods have been shown to induce DNA damage, chromosomal damage and to inhibit DNA repair. Using the comet assay and the micronucleus test, we now investigated temperature- and time-related effects on DNA damage and chromosomal effects of hyperthermia on the A549 human lung cell line. We also related the genotoxic effects to cytotoxic effects and the induction of apoptosis. Our results indicate that exposure to hyperthermia (42-48°C for 30-120min) induced genotoxic effects in a temperature- and time-related manner. Interestingly, hyperthermia-induced DNA damage measured by the comet assay was not rapidly removed by post-incubation at 37°C but even increased after exposure to 48°C for 60min. Cytotoxic effects occurred in parallel to the genotoxic effects but apoptosis was not significantly induced under these experimental conditions.

Insensitivity of the in vitro cytokinesis-block micronucleus assay with human lymphocytes for the detection of DNA damage present at the start of the cell culture.

The cytokinesis-block micronucleus assay (CBMN assay) with cultured human lymphocytes is a well-established assay in genotoxicity testing and human biomonitoring. For both approaches, human lymphocytes are stimulated by phytohaemagglutinin (PHA) and cultured for about 72 h; 44 h after PHA stimulation, cytochalasin B (CytB) is added and micronuclei (MN) are scored in binucleated cells. The main difference between these two applications is the way lymphocytes are exposed to mutagens. In order to maximise the probability of detecting a mutagen, the OECD guideline 487 recommends starting the exposure to the test substance at 44-48 h after PHA stimulation. In human biomonitoring, blood samples are taken from subjects exposed to environmental mutagens in vivo and lymphocytes with induced DNA damage at the start of the cell culture are investigated with regard to potentially increased MN frequencies in binuclear lymphocytes. We compared the sensitivity of these two protocols by either treating lymphocyte cultures for 2h with known DNA-damaging mutagens at the start of the culture or 42 h after PHA stimulation. The mutagens used were methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-ethylurea (ethyl nitrosourea; ENU), styrene oxide (SO), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and mitomycin C (MMC). All substances induced MN under the conditions of the standard in vitro CBMN assay but only MMC clearly induced MN in lymphocytes exposed at the start of the culture. All mutagens (except MMC, a known crosslinker) were tested by the comet assay with blood cultures exposed at the start of the culture and clearly induced DNA migration. The nuclear division index (NDI) indicated that damaged lymphocytes proliferated well in these experiments. The lack of increased MN frequencies despite increased damage levels and good proliferation suggests that the CBMN assay is rather insensitive for the detection of mutagens/clastogens when damage is induced at the start of the blood cultures. Potential consequences for the interpretation of human biomonitoring studies are discussed in this article.

Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to formaldehyde.

A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May-Grünwald-Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.96 vs 0.81 MN per 1000 cells). The slides were coded before re-evaluation and the code was broken after the complete re-evaluation of the study. A total of 1000 binucleated cells (BNC) were analysed per subject and the frequency of MN (in ‰) was determined. Slides were distributed equally and randomly between two scorers, so that the scorers had no knowledge of the exposure status. Scorer 2 (32 exposed, 36 controls) measured increased MN frequencies in exposed workers (9.88 vs 6.81). Statistical analysis with the two-sample Wilcoxon test indicated that this difference was not significant (p=0.17). Scorer 3 (20 exposed, 46 controls) obtained a similar result, but slightly higher values for the comparison of exposed and controls (19.0 vs 12.89; p=0.089). Combining the results of the two scorers (13.38 vs 10.22), a significant difference between exposed and controls (p=0.028) was obtained when the stratified Wilcoxon test with the scorers as strata was applied. Interestingly, the re-evaluation of the slides led to clearly higher MN frequencies for exposed and controls compared with the first evaluation. Bland-Altman plots indicated that the agreement between the measurements of the different scorers was very poor, as shown by mean differences of 5.9 between scorer 1 and scorer 2 and 13.0 between scorer 1 and scorer 3. Calculation of the intra-class correlation coefficient (ICC) revealed that all scorer comparisons in this study were far from acceptable for the reliability of this assay. Possible implications for the use of the CBMNT in human biomonitoring studies are discussed.

Does formaldehyde induce aneuploidy?

Formaldehyde (FA) was tested for a potential aneugenic activity in mammalian cells. We employed tests to discriminate between aneugenic and clastogenic effects in accordance with international guidelines for genotoxicity testing. The cytokinesis-block micronucleus test (CBMNT) in combination with fluorescence in situ hybridisation (FISH) with a pan-centromeric probe was performed with cultured human lymphocytes and the human A549 lung cell line. FA induced micronuclei (MN) in binuclear cells of both cell types under standard in vitro test conditions following the OECD guideline 487. FISH analysis revealed that the vast majority of induced MN were centromere negative, thus indicating a clastogenic effect. A similar result was obtained for MN induced by γ-irradiation, whereas the typical aneugens colcemid (COL) and vincristine (VCR) predominantly induced centromere-positive MN. Furthermore, COL and VCR clearly enhanced the MN frequency in mononuclear lymphocytes in the CBMNT, whereas such an effect was not observed for γ-irradiation and FA. In experiments with the Chinese hamster V79 cell line, the aneugens COL and VCR clearly increased the frequency of tetraploid second division metaphases, whereas FA did not cause such an effect. Altogether, our results confirm the clastogenicity of FA in cultured mammalian cells but exclude a significant aneugenic activity.

Analysis of micronuclei, histopathological changes and cell proliferation in nasal epithelium cells of rats after exposure to formaldehyde by inhalation.

The frequencies of micronuclei (MN), histopathological changes and cell proliferation were determined in the nasal epithelium of male Fischer-344 rats after exposure to formaldehyde (FA) by whole-body inhalation for four weeks (6h/day, 5 days/week). Groups of 12 rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15ppm. The micronucleus test (MNT) was performed with nasal epithelial cells prepared from six animals per group. Two thousand cells per animal were analysed for the presence of MN. The other six rats per group were subcutaneously implanted with osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdUrd), three days prior to necropsy. Paraffin sections were made from the nasal cavity (four levels) of these animals for histopathology and cell-proliferation measurements. The frequency of cells with MN was not increased in any of the groups. However, there was also no induction of MN in nasal cells of rats exposed to a single dose of cyclophosphamide (CP, 20mg/kg) by gavage and analysed 3, 7, 14 or 28 days after the treatment. In contrast, nasal epithelial cells from rats exposed to 10 or 15ppm FA vapour showed clear site-specific pathological changes (focal epithelial degeneration, inflammation and squamous metaplasia) in a decreasing gradient (anterior to posterior). Analysis of slides after anti-BrdUrd antibody staining clearly indicated increased cell proliferation (unit length labelling indices, ULLI) after exposure to 6ppm and higher. No treatment-related effects were measured after exposure to 0.5, 1 and 2ppm. When comparing the cell-proliferation rate of normal epithelium with that of directly adjacent metaplastic epithelium, no consistent pattern was found: depending on the location, cell proliferation of normal epithelia was either higher or lower. Our results support previous findings demonstrating local cytotoxic effects in the nose of rats after inhalation of FA. However, induction of MN in the nasal epithelium as an indicator of a mutagenic effect was not seen. Because only limited experience exists for the MNT with rat nasal epithelial cells, this result has to be interpreted with great care. The contribution of mutagenicity to FA's carcinogenicity in rat nasal epithelium remains unclear.

Micronuclei in peripheral blood from patients after cytostatic therapy mainly arise ex vivo from persistent damage.

The micronucleus test (MNT) is a well-established assay in genotoxicity testing and human biomonitoring. The cytokinesis-block micronucleus test (CBMNT) is the preferred method for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. It is, however, unclear to what extent mutagen exposure either leads to the formation of MN already in vivo or to the formation of MN ex vivo during cell culture as a consequence of persisting DNA damage. To address this question, we investigated peripheral blood of 22 patients who had received cytostatic therapies including drugs with clastogenic and aneugenic effects. We also performed the MNT with blood samples from 13 healthy controls without relevant mutagen exposure. The incidence of MN was studied 24, 48 and 72 h after the start of the culture in mononuclear lymphocytes in cultures without cytochalasin B and also at 72 h in binucleated lymphocytes in the standard CBMNT. The mean frequency of binuclear cells with MN in the CBMNT was clearly increased in blood samples from patients (29.3 versus 10.2 per 1000 in controls). In contrast, mononuclear lymphocytes analysed 24 or 48 h after start of the cultures only revealed a marginal increase in MN frequencies in comparison to controls. These results suggest that mutagen exposure in vivo mainly leads to the formation of MN during ex vivo proliferation of lymphocytes as a consequence of persistent damage. Characterization of MN in binuclear lymphocytes from patients by fluorescence in situ hybridization (FISH) with a pan-centromeric probe indicated that MN arose by clastogenic and aneugenic mechanisms. A high portion of MN was relatively large and exhibited several centromere signals. If the results of this study with patients exposed to cytostatic drugs also apply to other kinds of mutagen exposure, increased MN frequencies in the CBMNT can only be expected for exposures leading to persistent damage in peripheral lymphocytes and MN formation during ex vivo lymphocyte culture.

Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells.

The alkaline comet assay was used to further characterize the induction of DNA-protein crosslinks (DPX) by formaldehyde (FA) and their removal in the human lung cell line A549 and in primary human nasal epithelial cells (HNEC). DPX were indirectly measured as the reduction of gamma ray-induced DNA migration. FA induced DPX in A549 cells in a concentration-related manner in the range of 100-300 microM. This result is in agreement with previous studies using different mammalian cell lines. The main new findings of the present study are: (i) Determination of cytotoxicity in relation to genotoxicity strongly depend on the method used. Cytotoxicity measured as the reduction in cell counts 48 hr after addition of FA to the cultures occurred parallel to the induction of DPX while colony forming ability was already reduced at 10 times lower FA concentrations; (ii) DPX induced by a 1-hr FA treatment were completely removed within 8 hr cultivation in fresh medium while in the presence of FA in the medium DPX levels remained unchanged for 24 hr; (iii) Induction and removal of DPX did not fundamentally differ between exponentially growing and confluent A549 cultures; (iv) Slowly proliferating HNEC showed the same sensitivity towards FA-induced DPX as A549 cells (i.e. the same FA concentrations induced DPX under the same experimental conditions) and removed DPX with a similar efficiency. In summary, these results contribute to a better understanding of the genotoxic activity of FA in vitro and indicate that the tested cultured primary and permanent human cells do not differ fundamentally with regard to the processing of FA-induced primary genotoxic effects.

The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity.

The DNA-replication inhibitors aphidicolin (APC) and hydroxyurea (HU) were tested for their ability to induce effects on DNA in the in vitro alkaline comet assay with V79 cells. APC concentrations up to 15 microM and HU concentrations up to 500 microM did not significantly increase the extent of DNA migration after treatment during 4h. Treatment for 18 h, however, led to inconsistently significant increase in DNA migration. These increases in DNA migration were accompanied by severe cell-cycle disturbances, cytotoxic effects (reduced population doubling and reduced mitotic index) and increased frequencies of cells with chromosome aberrations. The results indicate that substances with such secondary effects on DNA (in contrast to agents that directly damage DNA) only induce effects in the comet assay after prolonged exposure, together with cytotoxic effects. We conclude that slight inhibition of DNA replication and cell-cycle delay per se do not cause significant effects in the in vitro comet assay under standard test conditions. Furthermore, the in vitro comet assay seems to be less sensitive towards this type of secondary DNA effects than the in vitro chromosome aberration test.

Low sensitivity of the comet assay to detect acetaldehyde-induced genotoxicity.

Acetaldehyde (AA) is known to induce DNA-protein cross-links (DPX) and other genotoxic and mutagenic effects in cultured mammalian cells. Compared to formaldehyde (FA), AA is a very weak inducer of DPX and increased DPX levels are only measured at high, cytotoxic concentrations by different methods. Besides DPX, AA also induces DNA-DNA cross-links. Because the comet assay is increasingly used for the detection of cross-linking agents, we characterized the effects of AA in the comet assay in relation to cytotoxicity and other genetic endpoints such as the induction of sister chromatid exchange (SCE) and micronuclei (MN). The standard alkaline comet assay did not indicate induction of DNA strand-breaks by AA in a range of concentrations from 0.2 to 20 mM. AA at a concentration of 20 mM was clearly cytotoxic and reduced cell growth and population doubling to less than 50% of the control. Using the comet assay modification with proteinase K, slightly enhanced DNA migration was measured in comparison to treatment with AA only. No significant induction of cross-links by AA (measured as reduction of gamma ray-induced DNA migration) was determined by the comet assay. A small and reproducible but statistically not significant effect was measured for the AA concentration 20 mM. A clear and concentration-related increase in the frequency of sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (0.2 and 0.5 mM, respectively). These results suggest that the comet assay has a low sensitivity for the detection of AA-induced DNA lesions leading to the induction of SCE and MN.

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

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

Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in cultured mammalian cells are not independently reproducible.

Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Using the comet assay, the micronucleus test and the chromosome aberration test with human fibroblasts (ES1 cells), the EU-funded "REFLEX" project (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) reported clearly positive effects for various exposure conditions. Because of the ongoing discussion on the biological significance of the effects observed, it was the aim of the present study to independently repeat the results using the same cells, the same equipment and the same exposure conditions. We therefore exposed ES1 cells to RF-EMF (1800 MHz; SAR 2 W/kg, continuous wave with intermittent exposure) for different time periods and then performed the alkaline (pH>13) comet assay and the micronucleus test (MNT). For both tests, clearly negative results were obtained in independently repeated experiments. We also performed these experiments with V79 cells, a sensitive Chinese hamster cell line that is frequently used in genotoxicity testing, and also did not measure any genotoxic effect in the comet assay and the MNT. Appropriate measures of quality control were considered to exclude variations in the test performance, failure of the RF-EMF exposure or an evaluation bias. The reasons for the difference between the results reported by the REFLEX project and our experiments remain unclear.

Dysfunction of the MDM2/p53 axis is linked to premature aging.

The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient's primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation's aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.

Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15µM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

Enhancement of genotoxic effects in the comet assay with human blood samples by aphidicolin.

The comet assay (single cell gel electrophoresis) has become increasingly used in human biomonitoring. In its standard version at pH > 13, DNA lesions such as DNA double-strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. Besides DNA damage, strand break formation during excision repair can also increase DNA migration. Inhibitors of DNA repair have been shown to enhance the DNA effects of mutagens and the use of repair inhibitors has been proposed for human biomonitoring studies to increase the sensitivity of the comet assay. To further evaluate the usefulness of such an approach we performed an experimental study with human blood and tested the enhancing effect of aphidicolin (APC) on DNA effects induced by different mutagens. Our results clearly show that APC enhances the genotoxic effects of benzo[a]pyrene diolepoxide (BPDE), bischloroethylnitrosurea (BCNU) and methyl methanesulfonate (MMS), but has no significant effect on gamma radiation-induced DNA effects. The enhancing effect is seen in unstimulated and PHA-stimulated blood, indicating repair activity under both conditions but the effect is stronger in stimulated blood. Our results indicate that APC can be used to increase the sensitivity of the comet assay towards a broad spectrum of induced primary DNA lesions and support the usefulness of this approach. However, for human biomonitoring, a sensitive protocol still has to be established.

Oxygenated water does not induce genotoxic effects in the comet assay.

Drinking of oxygenated water (i.e. water with increased concentration of physically dissolved oxygen) is said to improve oxygen availability of the body and will do the consumer good. However, increased oxygen concentrations can also lead to an increased production of reactive oxygen species (ROS). If antioxidant defences are not completely efficient, ROS can cause cell injury including DNA damage. We therefore investigated whether drinking of oxygenated water can lead to increased DNA damage in peripheral blood cells of test subjects. We also tested whether direct exposure of V79 Chinese hamster cells to oxygenated medium or oxygenated Hank's solution for various time periods induces DNA damage. Induction of DNA damage was measured with the alkaline comet assay (single cell gel electrophoresis). The comet assay, in particular the modification with FPG post-treatment for the determination of oxidative DNA base damage, has been proven to be extremely sensitive for the detection of oxygen-induced DNA damage. However, both the in vivo and the in vitro studies with the comet assay in the absence and presence of FPG post-treatment did not provide evidence for a genotoxic effect of oxygenated water.

Sensitivity of the FPG protein towards alkylation damage in the comet assay.

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5-12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.

Mutagen sensitivity of peripheral blood from women carrying a BRCA1 or BRCA2 mutation.

We are studying the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and a heterozygous mutation in the breast cancer susceptibility genes BRCA1 or BRCA2. Besides various other functions, BRCA proteins seem to be involved in DNA repair processes like transcription-coupled and double-strand break (dsb) repair. Our previous results indicated a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei (MN) by gamma irradiation and hydrogen peroxide (H2O2). In contrast to the results with the micronucleus assay, we found no significant individual difference between women with and without a BRCA1 mutation with respect to the induction and repair of DNA damage in the alkaline comet assay. We now investigated further cases heterozygous for a BRCA1 mutation and cases heterozygous for a BRCA2 mutation and show that enhanced micronucleus formation after gamma irradiation and H2O2-treatment is also a feature of lymphocytes carrying a BRCA2 mutation. Investigations with the comet assay did not reveal clear differences with regard to the induction of DNA damage on the individual level. There were also no significant differences between blood samples carrying a BRCA1 or BRCA2 mutation and blood samples from normal controls when the repair capacities (i.e. the kinetics of the removal of radiation-induced DNA effects in the comet assay) were compared. Our results indicate that mutagen sensitivity of lymphocytes heterozygous for a BRCA2 mutation is similar to that of cells with a BRCA1 mutation and BRCA1 and BRCA2 cannot be differentiated at present with the micronucleus test (MNT) or the comet assay.

Characterization of the genotoxic potential of formaldehyde in V79 cells.

Formaldehyde (FA) is known to be genotoxic and mutagenic in proliferating mammalian cells in vitro. The present study was performed to further characterize its genotoxic potential in the V79 Chinese hamster cell line. The induction of DNA strand breaks and DNA-protein cross-links (DPXs) was measured by the comet assay in relationship to the induction of sister chromatid exchanges (SCEs) and micronuclei (MN). Induction of DNA strand breaks was found neither with the standard protocol of the alkaline comet assay nor with modifications using extended electrophoresis times or proteinase K. The concentration-effect relationship for the genotoxic effects was characterized by fitting different curves to the data. A two-phase regression model fitted best in comparison with a linear or a quadratic model and indicated practical thresholds for the induction of SCE and MN. For the induction of DPX as measured by the comet assay, neither a linear concentration-response relationship nor any of the tested models fitted well to the data. Three repeated treatments with genotoxic concentrations of FA with a 3-h interval led to enhanced levels of DPX and MN while the same treatments with a 24-h interval did not enhance FA genotoxicity but suggested adaptive protection against the DNA-damaging action of FA.