Mutagenesis Advance Access originally published online on September 13, 2007
Mutagenesis 2007 22(6):387-394; doi:10.1093/mutage/gem031
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Characterization of the genotoxic potential of formaldehyde in V79 cells
Universität Ulm, Institut für Humangenetik, D 89069 Ulm, Germany
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.
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Introduction |
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Formaldehyde (FA) is highly reactive, induces cytotoxic and genotoxic effects and there is evidence in experimental animals and in humans for its carcinogenicity (1
). Previous investigations have already demonstrated the genotoxic potential of FA in proliferating cultured mammalian cell lines (1,2). It is generally accepted that the primary DNA damage induced by FA is a DNA–protein cross-link (DPX). FA-induced DPXs are removed by spontaneous hydrolysis and active repair in different cell types with half-lives between 12 and 18 h (3–6). Unrepaired DPX can arrest DNA replication and lead to the induction of other genotoxic effects such as sister chromatid exchanges (SCEs) during replication (3,4). Incomplete repair of DPX can lead to the formation of mutations (7) and chromosomal effects such as micronuclei (MN) seem to be most efficiently induced (3,8). Besides DPX, FA also induces hydroxymethyl adducts in DNA (9,10) but the relevance of these DNA modifications for FA-induced mutagenicity is unclear. While the majority of studies using ‘strand break assays’ such as alkaline elution or the comet assay only found the cross-linking effect of FA, some studies also reported the induction of DNA strand breaks by FA (11,12). There are indications that DNA strand breaks are induced by FA at low concentrations while DPXs are measured at higher concentrations (12) but the biological basis of strand break formation and the genetic significance of such effects are still unknown. FA is a naturally occurring biological compound that is present in cells and body fluids in most living organisms. Physiological amounts of FA are endogenously formed from serine, glycine, methionine and choline by demethylation of N-, O- and S-methyl compounds. Exogenous FA is rapidly metabolized after absorption. FA—either endogenously formed or from exogenous exposure—can undergo several possible metabolic pathways. Detoxification rapidly occurs via the multi-step pathway yielding formate and CO2. FA dehydrogenase (FAD) and other enzymes involved in this pathway seem to be ubiquitous enzymes and also glutathione (GSH) as a cofactor of FAD is ubiquitously present.
Humans are exposed to FA at the workplace usually at low concentrations but frequently repeatedly for longer time periods. This might lead to an accumulation of DNA damage and increase the risk for the formation of mutations. However, accumulation of DPX was not found in the nasal respiratory mucosa of rats exposed to FA for 12 weeks at airborne concentrations of 6 or 10 ppm (13). An earlier in vitro mutation study showed that multiple FA treatments resulted in increased mutation frequencies (TK mutations in TK6 cells) but the combined effect was less than a single treatment of equivalent concentration x time (14). This result suggests that even under mutagenic conditions, FA-induced DNA damage does not accumulate in mammalian cells.
It was the aim of the present study to further characterize the genotoxic action of FA with regard to the induction of DPX and other kinds of DNA lesions and to investigate the genetic consequences in relation to the formation of SCE and MN. Concentration–effect relationships were investigated by fitting different curves to the measurements of the genotoxicity tests to see whether a linear function fits the data adequately or whether a more complex model is more appropriate. Finally, experiments with repeated FA treatments were performed to better understand the relationship between the induction of DPX, DPX removal and the possibility of DPX accumulation.
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Materials and methods |
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Cell culture
V79 cells (a permanent Chinese hamster cell line) were cultivated in modified Eagle medium supplemented with 10% fetal calf serum (FCS) and antibiotics. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and harvested with 0.15% trypsin and 0.08% ethylenediaminetetraacetic acid (EDTA). For the experiments, cells were seeded into plastic flasks (T25) 24 h prior to FA exposure. If not otherwise stated, cells were treated with FA (CAS No. 50-00-0) in serum-free medium. Duration of treatment was 1 h for the induction of DPX and 2 h for the induction of SCE and MN. FA (16% ultrapure, methanol free) was bought from Polysciences, Inc., Warrington, PA, USA, and diluted in Hank's solution immediately before use. If not specifically indicated, the chemicals used in these experiments were purchased from Sigma (Munich, Germany). Cell culture media and ingredients were obtained from Invitrogen (Karlsruhe, Germany). Agarose (NEEO) was supplied by Roth (Karlsruhe, Germany) and low melting agarose (LMA, SeaPlaque, ‘GTG’) was from Biozym (Hameln, Germany).
Comet assay
The comet assay was performed according to a standard protocol which is usually used in our laboratory (15). Aliquots of 5 µl cell suspension (about 15 000 cells) were mixed with 120 µl low melting point agarose (0.5% in phosphate-buffered saline) and added to microscope slides (with frosted ends), which had been covered with a bottom layer of 1.5% agarose. Slides were lysed (pH 10; 4°C) for at least 1 h and processed as previously described in detail (15) using a time of alkali denaturation of 25 min and electrophoresis (0.86 V/cm) of 25 min at a pH >13. Deviations from this protocol are indicated when appropriate. Proteinase K (PK) treatment was performed after lysis (16). Slides were washed three times in Tris/EDTA (TE) buffer (100 µM Tris, 5 µM EDTA, pH 10), then covered with 100 µl PK (1 mg/ml TE buffer) and incubated for 2 h in a moist chamber at 37°C. Controls were incubated with 100 µl TE buffer only. After removing the coverslip, slides were processed as usual. Determination of DPX by the reduction of induced DNA migration was accomplished by irradiating FA-treated cultures with 2 Gy 
rays (Cs-137; Gammacell 2000, Nuclear Data, Germany). Slides were coded and images of 50 randomly selected cells stained with ethidium bromide (EtBr) were analysed from each slide. Measurements were made for 50 cells per slide by image analysis (Comet Assay II, Perceptive Instruments, Haverhill, UK). For all experiments, we evaluated four image analysis parameters: tail length, tail migration, tail intensity and tail moment (TM). In none of the experiments, there was a relevant difference between these parameters. Therefore, we chose one parameter (TM) for the presentation of the results. TM is calculated according to the formula: TM = (tail intensity/total comet intensity) x (tail centre of gravity – peak position).
Cell viability was determined at the end of the treatment (single and repeated treatments) using the fluorescein diacetate (FDA)/ethidium bromide assay. Two hundred cells were counted per data point and the percentage of dead cells (orange-stained nuclei) was determined.
SCE test
SCE tests were performed according to (3,16). After treatment with FA, the medium was changed and complete medium supplemented with 10 µg/ml 5-bromodeoxyuridine was added. Cells were cultivated for the duration of two cell cycles (about 24 h) and colcemid (2 x 10–7 M) was added for the final 2 h. Chromosome preparation was done following standard procedures. Cells were centrifuged, re-suspended in 0.4% KCl for 20 min and fixed three times in methanol:glacial acetic acid (3:1). For sister chromatid differentiation, air-dried slides were covered with Sörensen buffer (pH 6.8) and irradiated with an 8-W UV lamp (254 nm) at a distance of 10 cm for 20 min. Subsequently, slides were incubated in 1 x SSC for 30 min at 60°C and then stained with 5% Giemsa in Sörensen buffer. SCE was scored in 30 cells per sample from coded slides. Cytotoxicity was determined by scoring first division mitoses (M1), second division mitoses (M2) and third division mitoses (M3) among 100 metaphases and calculating the proliferation index (PI) according to the formula: PI = [M1 + (2 x M2) + (3 x M3)]/100.
Micronucleus test (MNT)
The MNT was performed as described earlier (3). V79 cells were cultivated after exposure for another 18 h. Cells were detached by trypsin, exposed briefly to a hypotonic solution (0.56% KCl) and fixed three times with methanol/glacial acetic acid (5 + 1). Air-dried slides were stained with acridine orange (60 µg/ml in phosphate buffer). The frequency of MN was determined by analysing 1000 cells from coded slides.
Statistical analysis
Pre-experiments were performed to define the appropriate FA concentrations for the various genotoxicity tests. Further, a simulation study was carried out to give guidance in choosing the number of replicates as well as the spacing of concentrations under various linear and non-linear dose–response models. After specification of expected response levels and their standard deviations, the computer program generated virtual experiments (runs) with random responses from which the F statistic for lack of fit (17) was determined in each run. The F test for lack of fit was used to test the hypothesis whether the concentration–effect relationships in the genotoxicity tests are appropriately described by a linear function (null hypothesis: the effect is a linear function of the concentration, i.e. low P-values indicate that a more complex model should be fitted). This statistical test was applied in four versions: [1] based on the appropriate sums of squares from simple linear regression; [2] based on a regression analysis treating the measurements under concentration 0 (null value) as a covariate; [3] based on weighted regression analysis, where weighting was done using the reciprocal of the variance of the measurements at different concentrations and [4] under both [2] and [3]. The rationale behind [2] was the adjustment of measurements from different experiments. In this model, we consider each of the five experiments (i.e. the determination of responses in a series including all concentrations) as a block in the design and treat the control value (concentration zero) of the experiment as a covariate. In practice, we apply the test for lack of fit to the residual measurements after removing the impact of the particular experiment. Weighted regression analysis [3] was applied to cope with varying dispersion at different concentrations. All concentration–response modeling was done using the concentration in micromolar as the concentration metric.
It should be noted that lack of fit for a purely linear model does not exclude that in certain ranges the concentration–response relationship is linear or may be well approximated by a linear curve. Hence, using regression analysis, we also checked whether the concentration–effect relationships contain statistically significant linear components.
For description of concentration–effect relationships, the fitting of three different curves to the measurements was attempted: (A) a linear curve for comparison of fit, (B) a two-phase piecewise linear regression curve incorporating an implicit threshold and (C) a quadratic function without threshold. Model (A) contains two unknown parameters a and b (expected response = a * concentration + b), model (B) contains three parameters a, b and d (expected response = a + b * d, if concentration 
d; response = a + b * concentration, if concentration >d) and model (C) has three as well (expected response = a + b * concentration + c * concentration2). The piecewise linear curve was constructed using a simplex procedure (implemented in SAS IML) under the conditions that in the first phase the curve is a straight line parallel to the abscissa (indicating no increase of effect), whereas in the second phase it increases linearly. The concentration that separates the two phases may be considered as a threshold concentration for the conditions of the test. The parameters were estimated using the least square method. Goodness of fit was assessed by the residual sum of squares (RSSs) and the Akaike information criterion (AIC) according to the formula AIC = 2k + n * ln (RSS/n) under normal distribution of the responses (k = number of parameters and n = number of observations). Note that AIC includes a penalty for adding model parameters.
For the determination of concentration–effect relationships, five independent experiments were carried out under the same conditions. The other experiments (figures 1, 2, 9 and 10) were independently performed three times. Each independent experiment was performed at different days with freshly prepared FA solutions with parallel subcultures for each of the FA concentration tested and the appropriate controls. Comparisons between the mean responses of the different concentrations and the mean control value were carried out under analysis of variance models taking into consideration the experimental design. A statistically significant difference was set at P < 0.05.
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Results |
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Induction of DNA effects in the comet assay
Figure 1 shows the effects of FA (1 h treatment in serum-free medium) in the comet assay with V79 cells under standard conditions (25 min alkaline treatment, 25 min electrophoresis; left) and under conditions with extended electrophoresis time (35 min). It can be seen that there is no significant effect under standard conditions over a wide range of FA concentrations (0.001–200 µM). Increasing the time for electrophoresis led to increased DNA migration (TM) in control cultures. FA in the range of 0.001–1 µM did not exert a significant effect on DNA migration, in particular there was no increase in DNA migration (i.e. no indication for a strand-breaking effect). Under both conditions, there is a trend towards lower TM values with increasing FA concentrations. With an electrophoresis time of 35 min, a significant decrease in DNA migration was measured (indicative for DPX) for FA concentrations of 10 µM and higher up. The fluorochrome-mediated viability test indicated only 1–3% dead cells in these experiments, thus demonstrating that the comet assay results were not significantly influenced by dead cells.
PK has been used previously to differentiate between DPX and DNA–DNA cross-links (16) or for the determination of DPX (18). We used post-treatment of slides with PK under the standard conditions of the comet assay (25 min electrophoresis) to see whether DNA strand breaks may be hidden behind the cross-linking effect of FA. Post-treatment with PK slightly enhanced DNA migration in controls and FA-treated cultures and abolished the cross-linking effect of FA (Figure 2). An increase in DNA migration a little above control level was also observed previously in a comet assay with FA and PK (18). However, the procedure as such (i.e. treatment with buffer only) also had a slight effect and increased assay variability and the effect of PK treatment was not statistically significant compared to the control with buffer (b).
Concentration–effect relationship of FA-induced DPX
DPXs were measured by the reduction of gamma-irradiation induced DNA migration in the comet assay (3). Following FA treatment, cells were exposed to 2 Gy gamma irradiation and then analysed in the alkaline (pH 13) comet assay. Figure 3A shows that FA reduced DNA migration in the comet assay in a concentration-related manner (mean values of five independent experiments). Clear effects (P < 0.01) are seen after treatment with 25 µM and above in accordance with earlier studies (3). A steep decrease is seen at the concentration 200 µM. A second independent series of five experiments were performed in the low concentration range (1–10 µM). The results are shown in Figure 3B and clearly indicate that there is no genotoxic effect of FA in the comet assay in this range of concentration. 100 µM FA was also tested in these experiments (as a positive control) and led to significant reduction in DNA migration compared to the irradiated control culture (P < 0.01).
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Both presence of a linear component in the concentration–effect relationship and the adequacy of a linear model to describe the data were checked using four different approaches. The corresponding P-values for each of the tests are summarized in Table I. With regard to the effects in the comet assay (Figure 3A), the results clearly demonstrate a linear component but a more complex model than a linear one seems to provide improved fit. Hence for comparison, exemplarily three different curves were considered to describe the measurements: a linear function, a quadratic function as well as a two-phase piecewise regression curve (see Materials and methods). Figure 4 shows that the linear function and the quadratic function did not completely describe the concentration–response relationship (i.e. the data points and the curves do not agree). It was impossible to meaningfully fit a two-phase piecewise regression curve to the TM values (data not shown).
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In the second set of comet assay experiments (concentrations 1–10 µM; the concentration 100 µM was included as a positive control), FA did not induce detectable DPX in this range of concentrations (P = 0.93). Although it was not possible to exactly determine a practical threshold for the DPX-inducing effect from these experiments, the data suggest a practical threshold between 10 and 100 µM.
Concentration–effect relationship for FA-induced SCE
SCEs were induced by FA in V79 cells at a concentration of 100 µM and above in accordance with our earlier results (3). The genotoxic effect occurred in parallel to the cytotoxic effect measured by the reduction of the proliferation index (Figure 5). Although there is strong evidence for a linear component, the statistical tests under various underlying models (Table I) indicate that the relationship cannot be completely described by a linear curve and extra terms might provide a better fit. When fitting the three different curves (a linear function, a quadratic function and a two-phase piecewise regression curve) to the measurements and assessing goodness of fit by the RSSs and AIC, we find for the SCE test: 44.8 and 13.4 (linear; predicted response = 4.07 + 0.068 * concentration), 21.3 and –9.9 (quadratic; predicted response = 4.81 + 0.023 * concentration + 0.00023 * concentration2) and 18.0 and –15.6 (two phase; predicted response = 5.11, if concentration 41, predicted response = 1.62 + 0.085 * concentration, if concentration >41). This result shows that the two-phase regression model fits best (lowest value) among the three. A comparison between the linear and the two-phase curve is shown in Figure 6 (the quadratic curve was omitted for reasons of clarity). A practical threshold for the induction of SCE under these experimental conditions can be assumed for a concentration between 25 and 50 µM.
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Concentration–effect relationship for FA-induced MN
In contrast to our earlier results (3
), the MNT was slightly more sensitive than the SCE test (Figure 7). A slight but statistically significant (P < 0.05) increase in the MN frequency was already determined for the concentrations 75 and 100 µM. Appropriate fit of a linear concentration–effect curve could only be rejected when the varying dispersion of the data was considered (weighted regression, Table I). Similar to the results of the SCE test, fitting the MNT measurements also showed that the piecewise regression model gives the best fit. Goodness of fit revealed the following RSSs and AIC for the MNT: 36.3 and –18.4 (linear; predicted response = 0.40 + 0.024 * concentration), 32.4 and –23.1 (quadratic; predicted response = 0.61 + 0.017 * concentration + 0.00002 * concentration2) and 28.2 and –30.7 (two phase; predicted response = 0.89, if concentration 45, predicted response = –0.33 + 0.027 * concentration, if concentration >45). A comparison between the linear and the two-phase curve is shown in Figure 8 (the quadratic curve was omitted for reasons of clarity). A practical threshold for the induction of MN can be assumed under these experimental conditions for a concentration between 25 and 50 µM which is very similar to the effect in the SCE test.
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Induction of DPX and MN after repeated treatments
In a further series of experiments, V79 cells were treated three times with FA with time intervals of 3 and 24 h, respectively. Cells were treated each time in complete medium (with serum) with the concentrations indicated and FA was not removed. Figure 9 summarizes comet assay results after treatment of V79 cells with FA for three times. The comet assay was always performed 1 h after the last treatment directly after irradiation with 2 Gy. A single FA treatment for 1 h in combination with 2 Gy irradiation was performed as a reference. DPXs are indirectly measured as a reduction in irradiation-induced DNA migration. Three treatments with 3-h time intervals caused enhanced cross-linking effects in the comet assay. In contrast, no significant cross-linking effect of FA was measured after (a third) FA treatment in cells pre-treated with FA for two times with 24-h intervals. The FDA/EtBr assay measured more than 97% viable cells in all of these experiments, thus indicating that the comet assay results were not significantly influenced by the presence of dead cells.
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Figure 10 summarizes MNT results after treatment of V79 cells with FA for three times with time intervals of 3 and 24 h, respectively. The MNT was always performed 18 h after the start of the last treatment, i.e. a time period sufficient to cover the time period of one and a half cell cycles. An experiment with a single treatment for 18 h was included as a reference. Three treatments with 3-h time intervals caused enhanced effects in the MNT. The cytotoxic effect of FA was also enhanced under these conditions and, therefore, the higher concentrations (75 and 100 µM) could not be evaluated due to the inhibition of proliferation. The cytotoxic effect was independently confirmed by parallel cell counts at the end of the culture period in these experiments (data not shown). Three repeated treatments with intervals of 24 h did not lead to enhanced effects compared to a single treatment; the MN frequencies were even a little bit lower after three FA exposures.
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Discussion |
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The genotoxicity of FA in cultured mammalian cells has been reported in numerous previous studies and reviewed by different expert groups (1
,2). It seems to be clear that FA induces DPX in proliferating and non-proliferating cells. Persisting DPXs lead to other genotoxic effects in proliferating cells such as SCE and also to mutations on the gene and chromosome level. Published data suggest that chromosomal effects (chromosome aberrations and MN) are predominantly induced while true gene mutations play a minor role (3,8). Despite the broad existing database, there are still open questions with regard to hazard characterization and risk assessment. Using three sensitive and relevant genotoxicity tests, namely the comet assay, the SCE test and the MNT, we now addressed three aspects of FA genotoxicity (i) the contribution of DNA strand breaks to FA genotoxicity, (ii) the concentration–effect relationship for the three genetic endpoints and (iii) the consequences of repeated FA exposures.
Our results indicate that FA does not significantly induce DNA strand breaks and that DPX seem to be the most relevant DNA damage after FA exposure. The alkaline comet assay is a very sensitive tool for the detection of DNA strand breaks. However, in the case of FA, DNA migration induced by strand breaks in the comet assay may be inhibited and masked under the standard conditions of the test by concurrently induced DPX. PK resolves DPX and thus should enable DNA migration due to strand breaks (16,18). However, the use of this comet assay modification actually abolished FA-induced inhibition of DNA migration but the effects did not significantly exceed the baseline levels. In particular, a strand-breaking effect of low FA concentrations (12) could not be confirmed. The reason why others found induction of strand breaks after FA exposure is unclear. The effect could be due to increased excision repair activity after FA exposure. At present, there is no satisfying explanation for the conflicting results but our thorough investigation suggests that DNA strand breaks do not play an important role for hazard characterization of FA.
Although it is often taken as axiomatic that mutagens/genotoxic carcinogens induce DNA damage at any level of exposure (linear concentration–response relationship), the possibility that even direct-acting DNA-reactive agents may have thresholded dose–responses is increasingly supported by experimental data (19). Various mathematical models exist to describe a concentration/dose–response relationship and to try to identify thresholds [for a review, see ref. (20)]. Our attempts to characterize the concentration–effect relationship of FA-induced genotoxicity and mutagenicity allow some important conclusions. Although there is clear evidence for a linear component in the concentration–effect relationship and linearity may be assumed as a first approximation, none of the investigated genotoxic effects could be completely described by a linear concentration–effect curve and the inclusion of extra terms provided a better fit. For SCE and MN, the two-phase regression model with lack of effect in the first phase gave the best fit to the measurements among these models and insinuated that a threshold concentration exists. In contrast, the induction of DPX indirectly measured by the comet assay could not be shown to comply with an easy-to-interpret regression model. Of course, rather complicated concentration–effect functions may be fitted, however, without theoretical rationale. The impossibility of modeling an appropriate concentration–effect relationship for FA-induced DPX is most likely not due to the nature of the genotoxic effect studied but can be explained by the high assay variability of the comet assay. In contrast to the other genotoxicity tests used, a positive effect in the comet assay strongly depends on the specific test conditions applied and can be influenced by a variety of test variables (15). A threshold for FA-induced genotoxicity might be explained by the high reactivity of FA and the possibility that low FA concentrations are inactivated by reactions with cellular components and do not reach the nuclear DNA. FA-induced DPXs were previously measured in an in vivo study in the nasal mucosa of rats exposed to a wide range of FA concentrations (0.3–10 ppm) for 6 h. The fitted concentration–response curve indicated a nonlinear formation of DPX. However, as DNA–protein cross-linking occurred at all concentrations tested, a threshold could not be determined (21).
Under the experimental conditions used here, a practical threshold for the induction of SCE and MN in V79 cells can be derived at a concentration between 25 and 50 µM. While DPXs are already induced at non-toxic concentrations, their transformation into SCE and MN obviously requires high FA concentrations which also cause cytotoxicity. It can be assumed that DNA repair significantly contributes to thresholds for genotoxicity/mutagenicity (19). Our data support the view that FA-induced SCE and MN only occur when DNA repair processes are overloaded and cytotoxicity becomes obvious.
Humans are exposed to FA at the workplace usually at low concentrations but frequently repeatedly for longer time periods. This might lead to an accumulation of DNA damage and increase the risk for the formation of mutations. However, accumulation of DPX was not found in the nasal respiratory mucosa of rats exposed to FA for 12 weeks at airborne concentrations of 6 or 10 ppm (13). In our experiments with proliferating V79 cells, the genotoxic effect of repeated exposures to FA clearly depended on the time schedule of the treatment. Short intervals (3 h) between the treatments enhanced the cytotoxic and genotoxic effects of FA while such an effect was not observed after longer intervals (24 h). These results are in accordance with our previous data regarding the removal of FA-induced DPX in V79 cells (3). Three hours after the end of the FA treatment, considerable amounts of residual DPX were measured, whereas DPXs were completely removed after 24 h. This means that accumulation of DPX and enhanced genotoxicity may occur after repeated exposures with short time intervals at a concentration/dose that causes enough persisting DPXs. Exposure to low FA concentrations (10 µM) and/or longer time intervals between exposures did not enhance FA genotoxicity. The results from the MNT confirm the findings in the comet assay. Genotoxicity and cytotoxicity is strongly enhanced only after repeated treatments with short intervals. It is unclear whether the effects in the MNT after repeated treatments with 24-h intervals are due to DPX induced in the last treatment or due to a delayed effect of the earlier treatments and some kind of adaptive protection against the induction of DPX. The comet assay results might suggest an adaptive response. This interpretation is also supported by an earlier in vitro mutation study with TK6 cells (14). Multiple FA treatments resulted in increased mutation frequencies at the TK locus but the combined effect was less than a single treatment of equivalent concentration x time. A biological mechanism underlying adaptive protection against FA-induced genotoxicity is not known. One might speculate that enzymes responsible for the detoxification of FA are inducible but there is at presence no evidence for such a regulation in mammalian cells (1). Further studies are in progress to show whether mammalian cells are actually able to adapt against the genotoxic action of FA.
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Funding |
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European Chemical Industry Council.
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Acknowledgments |
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The financial support is gratefully acknowledged. However, the views expressed in this publication are those of the authors and there is no conflict of interest in performing this study and presenting the results.
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* To whom correspondence should be addressed. Tel: +49 731 500 65440; Fax: +49 731 500 65402; Email: guenter.speit{at}uni-ulm.de
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1. International Agency for Research on Cancer (2006) IARC Monographs Vol. 88: Formaldehyde, 2-Butoxyethanol and 1-tert-Butoxypropan-2-ol. Geneva: WHO Press.
2. BfR (Federal Institute for Risk Assessment) (2006) Assessment of the Carcinogenicity of Formaldehyde [CAS No. 50-00-0]. (http://www.bfr.bund.de/cm/238/assessment_of_the_carcinogenicity_of_formaldehyde.pdf—accessed March 2007).
3. Merk O, Speit G. Significance of formaldehyde-induced DNA-protein crosslinks for mutagenesis. Environ. Mol. Mutagen. (1998) 32:260–268.[CrossRef][Web of Science][Medline]
4. Schmid O, Speit G. Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies. Mutagenesis (2007) 22:69–74.
5. Speit G, Schutz P, Merk O. Induction and repair of formaldehyde-induced DNA-protein crosslinks in repair-deficient human cell lines. Mutagenesis (2000) 15:85–90.
6. Quievryn G, Zhitkovich A. Loss of DNA-protein crosslinks from formaldehyde-exposed cells occurs through spontaneous hydrolysis and an active repair process linked to proteosome function. Carcinogenesis (2000) 21:1573–1580.
7. Barker S, Weinfeld M, Murray D. DNA-protein crosslinks: their induction, repair, and biological consequences. Mutat. Res. (2005) 589:111–135.[CrossRef][Web of Science][Medline]
8. Speit G, Merk O. Evaluation of mutagenic effects of formaldehyde in vitro: detection of crosslinks and mutations in mouse lymphoma cells. Mutagenesis (2002) 17:183–187.
9. Fennell TR. Development of methods for measuring biological markers of formaldehyde exposure. Res. Rep. Health Eff. Inst. (1994) 67:1–20.[Medline]
10. Zhong W, Que Hee SS. Formaldehyde-induced DNA adducts as biomarkers of in vitro human nasal epithelial cell exposure to formaldehyde. Mutat. Res. (2004) 563:13–24.[Web of Science][Medline]
11. Grafstrom RC, Fornace A Jr, Harris CC. Repair of DNA damage caused by formaldehyde in human cells. Cancer Res. (1984) 44:4323–4327.
12. Liu Y, Li CM, Lu Z, Ding S, Yang X, Mo J. Studies on formation and repair of formaldehyde-damaged DNA by detection of DNA-protein crosslinks and DNA breaks. Front. Biosci. (2006) 11:991–997.[Web of Science][Medline]
13. Casanova M, Morgan KT, Gross EA, Moss OR, Heck HA. DNA-protein cross-links and cell replication at specific sites in the nose of F344 rats exposed subchronically to formaldehyde. Fundam. Appl. Toxicol. (1994) 23:525–536.[CrossRef][Web of Science][Medline]
14. Craft TR, Bermudez E, Skopek TR. Formaldehyde mutagenesis and formation of DNA-protein crosslinks in human lymphoblasts in vitro. Mutat. Res. (1987) 176:147–155.[Web of Science][Medline]
15. Speit G, Hartmann A. The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair. Methods Mol. Biol. (2006) 314:275–286.[Medline]
16. Merk O, Speit G. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. Environ. Mol. Mutagen. (1999) 33:167–172.[CrossRef][Web of Science][Medline]
17. Neter J, Kutner MH, Nachtsheim CJ, Wassermann W. Diagnostics and Remedial Measures (1996) 4th edn. 115–124.
18. Hu Y, Kabler SL, Tennant AH, Townsend AJ, Kligerman AD. Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene. Mutat. Res. (2006) 607:231–239.[Web of Science][Medline]
19. Jenkins GJ, Doak SH, Johnson GE, Quick E, Waters EM, Parry JM. Do dose response thresholds exist for genotoxic alkylating agents? Mutagenesis (2005) 20:389–398.
20. Lovell DP. Dose-response and threshold-mediated mechanisms in mutagenesis: statistical models and study design. Mutat. Res. (2000) 464:87–95.[Web of Science][Medline]
21. Casanova M, Deyo DF, Heck HD. Covalent binding of inhaled formaldehyde to DNA in the nasal mucosa of Fischer 344 rats: analysis of formaldehyde and DNA by high-performance liquid chromatography and provisional pharmacokinetic interpretation. Fundam. Appl. Toxicol. (1989) 12:397–417.[CrossRef][Web of Science][Medline]
Received on March 19, 2007; revised on July 16, 2007; accepted on July 17, 2007.
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