Mutagenesis Advance Access originally published online on May 13, 2008
Mutagenesis 2008 23(5):355-357; doi:10.1093/mutage/gen025
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Further characterization of the genotoxicity of formaldehyde in vitro by the sister chromatid exchange test and co-cultivation experiments
Institut für Humangenetik, Universität Ulm, D-89069 Ulm, Germany
The induction of sister chromatid exchanges (SCE) was used to further characterize the genotoxic action of formaldehyde (FA) on cultured mammalian cells. FA induced SCE in V79 Chinese hamster cells and A549 human lung cells in a concentration-related manner. Addition of 5-bromodeoxyuridine (BrdUrd) for the differentiation of sister chromatids to visualize SCE 4 h after the FA treatment led to a clearly reduced induction of SCE in agreement with the repair kinetics of FA-induced DNA–protein cross-links. When A549 cells were treated with FA for 1 h and then co-cultivated with V79 cells in the presence of BrdUrd, a clear induction of SCE was measured in V79 cells. When the same experiment was performed including washing and change of medium after the FA treatment, no induction of SCE was measured in V79 cells. These results indicate that reactive FA remains in the cell culture medium for a longer time period despite the high reactivity of FA with macromolecules. However, FA that has entered a cell is not released and does not damage other cells. Possible implications for the mutagenicity of FA in vivo will be discussed.
 |
Introduction |
|---|
 Top Introduction Materials and methodsResults and discussionFundingReferences |
|---|
The in vitro genotoxicity and mutagenicity of formaldehyde (FA) have been demonstrated by various tests with cultivated mammalian cells (1
). Among other genetic end points, FA efficiently induced sister chromatid exchanges (SCE) in various cell culture systems (2–6). The mechanism of SCE formation is still not completely understood (7) but it is clear that SCE measured in second division metaphases in the SCE test occur during replication at stalled replication forks in the presence of 5-bromodeoxyuridine (BrdUrd). We have recently shown that FA treatment of human blood samples ex vivo only caused increased SCE frequencies in cultured lymphocytes when FA-induced DNA–protein cross-links (DPX) (measured by the comet assay) persisted until S-phase (5). Thus, SCE seem to be a sensitive and reliable measure of persisting DNA alterations induced by FA.
FA is a naturally occurring biological compound that is present in the cells in most living organisms. Exogenous FA rapidly binds to glutathione (GSH) and proteins and is efficiently 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. Formaldehyde dehydrogenase (FDH) and other enzymes involved in this pathway seem to be ubiquitous enzymes and also GSH as a cofactor of FDH is ubiquitously present. Due to its high reactivity, the half-life of FA in cellular systems is generally assumed to be very short and its half-life in blood has been determined to be 
1.5 min (8). However, recent results from our group suggested that FA continues to be active in cell cultures for several hours (9). Because it is of fundamental importance for the characterization of the genotoxic and mutagenic potential of FA to know whether the genotoxic activity is due to FA remaining in the cell culture medium or whether reactive FA can be passed from one cell to another, we designed co-cultivation experiments to address this question. We exposed human A549 cells to FA, co-cultivated them after exposure (with and without changing the exposure medium) with V79 Chinese hamster cells and measured the frequency of SCE in second division V79 metaphases. Due to the clearly distinguishable karyotypes, V79 cells can unequivocally be recognized and evaluated.
|
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResults and discussionFundingReferences |
|---|
Cell culture
V79 cells (a permanent Chinese hamster cell line) and A549 cells (an epithelial-like human lung cell line) were cultivated in minimal essential medium supplemented with 10% foetal 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. For the experiments, cells were seeded into plastic flasks (T12.5) 24 h prior to the start of the experiment. Cells were either treated with FA in medium without FCS (treatment for 1 h) or in complete medium (continuous treatment). FA (CAS No. 50-00-0; 16%, ultrapure, methanol free, FW 30.03) was supplied from Polysciences, Inc. (Warrington, PA, USA) and diluted in Hank's solution immediately before use. If not specifically indicated, the other chemicals used in these experiments were purchased from Sigma (Munich, Germany). Cell culture media and ingredients were obtained from Invitrogen (Karlsruhe, Germany).
SCE test
SCE tests were performed according to Speit et al. (9). In the standard protocol, BrdUrd dissolved in Hank's solution was added (final concentration 20 µg/ml) after treatment with FA and cells were cultivated for the duration of about two cell cycles (24 and 48 h for V79 and A549 cells, respectively). After addition of BrdUrd, cultures were protected from light. For the co-cultivation experiments, V79 cells were trypsinized and added (250 000 cells each) to the A549 cell cultures. V79 cells and BrdUrd were added either directly to the A549 cell cultures 1 h after the start of the FA treatment or the exposure medium was removed, the A549 cultures were washed with Hank's solution and fresh, BrdUrd-containing medium was added together with the V79 cells. Co-cultures of A549 and V79 cells were further kept in the incubator for 24 or 32 h.
To arrest cells in mitosis, colcemid (0.2 µg/ml) 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 30 min. Subsequently, slides were incubated in 2x SSC for 20 min at 58°C and then stained with 7% Giemsa in Sörensen buffer. SCE were scored in 30 metaphases per sample from coded slides.
Statistical analysis
The experiments were independently performed three times under the same conditions. Differences between the mean values of the independently repeated experiments were tested for significance using Student's t-test. A statistically significant difference was set at P < 0.05. The P-values were not adjusted for multiple testing.
|
Results and discussion |
|---|
|
TopIntroductionMaterials and methodsResults and discussionFundingReferences |
|---|
Induction of SCE in A549 cells and V79 cells
Figure 1 shows the induction of SCE by FA in A549 cells after treatment for 1 h and further cultivation in fresh, BrdUrd-containing medium for the duration of two cell cycles. SCE are induced in a concentration-related manner with a statistically significant increase over the control being measured at an FA concentration of 100 µM. This result perfectly corresponds to the induction of DPX, the primary FA-induced DNA lesions, determined by a modified comet assay in A549 cells (9
). Thus, the result of the SCE test demonstrates that SCE are a sensitive indicator for the determination of FA-induced genotoxicity. SCE are induced in V79 cells by FA in the same range of concentrations but the SCE-inducing effect is a little bit more pronounced (Figure 2, black bars). This can be due to the shorter cell cycle and higher replicative activity of V79 cells. These results confirm our previous findings (4,6) and demonstrate that the SCE test with V79 cells is highly reproducible and reliable. If V79 cells are exposed to FA for 1 h and BrdUrd is added 4 h after the medium was exchanged, induction of SCE in second division metaphases (cultivated for 24 h in the presence of BrdUrd) is clearly diminished (Figure 2, white bars). A slight and statistically significant increase in the frequency of SCE is seen at FA concentrations of 200 µM and higher. This result indicates that in the course of 4 h between the end of the exposure and the addition of BrdUrd, a relevant amount of SCE-inducing lesions is repaired. This finding corresponds to our earlier comet assay results, which showed that a significant portion of FA-induced DPX is removed from V79 cells within 4 h (4). Taken together, these experiments clearly confirm that SCE are a sensitive and reliable indicator for FA-induced DNA damage in proliferating cells. The correspondence with previously published comet assay results and the mechanism of SCE formation suggest that FA-induced DPX might be the relevant lesion for SCE induction.
|
|
Induction of SCE in co-cultivation experiments
When V79 cells and BrdUrd are added to A549 cells that were exposed to FA for 1 h, a clear and concentration-related increase in the SCE frequency was measured in V79 metaphases after co-cultivation for two cell cycles (Figure 3). This result indicates that 1 h after addition to an A549 cell culture there is still enough reactive FA available to induce SCE in V79 cells. The induction of SCE in V79 cells is similar to the effect measured in directly exposed V79 cells (Figure 2) but freshly trypsinized V79 cells exposed to FA seem to exhibit a higher vulnerability than pre-cultivated cells, leading to an increased frequency of FA-induced SCE. Thus, a direct comparison of the induced SCE frequencies may be limited. In any case, this experiment clearly demonstrates that in mammalian cell cultures, FA is not inactivated within 1 h but keeps its genotoxic potential and induces damage in previously unexposed cells. This result supports our recent findings with A549 cells that revealed a clear difference in the efficiency of DPX removal when FA remained in the cell culture medium (9
).
|
Figure 4 summarizes the results of the second co-cultivation protocol. The medium was changed and the A549 cultures were washed before the trypsinized V79 cells were added with fresh, BrdUrd-containing medium. There was no induction of SCE in the V79 cells after two cell cycles of co-cultivation even after co-cultivation with A549 cells exposed to a high (300 µM) concentration of FA. This result indicates that FA taken up by A549 cells at concentrations that clearly induce genotoxicity in these cells under these experimental conditions (see Figure 1) is not released from the A549 cells in an active form that is able to enter other cells and induce DNA damage there. FA seems to be bound and/or inactivated in A549 cells and there seems to be no release into the medium or exchange between cells. V79 cells attached within 1 h to the surface of the cell culture flask and were in direct contact with A549 cells. The experiments performed with directly FA-exposed V79 cells (Figure 2) or with V79 cells exposed to the treatment medium of A549 cells 1 h after the start of the exposure (Figure 3) clearly demonstrate that much lower FA concentrations are able to induce SCE.
|
Although it cannot be excluded that minor exchange of reactive FA may occur, a relevant genetic effect in cells not in first contact with FA is unlikely. As A549 cells and primary human nasal epithelial cells do not differ significantly with regard to their sensitivity towards FA-induced genotoxicity (9
), our findings may be of relevance for the in vivo situation. FA is well characterized as a local genotoxin/mutagen that induces a broad spectrum of genotoxic/mutagenic effects in directly exposed cells in vitro and at the site of first contact in vivo (1). It is still a matter of discussion, whether or not FA also induces systemic toxic/genotoxic effects although the abundance of evidence suggests that there is no delivery of inhaled FA to distant sites (10,11). If our findings also apply to the in vivo situation, FA would in fact only be genotoxic towards directly exposed cells and no transcellular transmission should be expected. Thus, our results support the view that FA, at least at the levels associated with occupational exposure, is unlikely to induce genotoxic effects away from the site of contact. |
Funding |
|---|
|
TopIntroductionMaterials and methodsResults and discussionFundingReferences |
|---|
European Chemical Industry Council (CEFIC).
|
Acknowledgments |
|---|
We wish to thank Mrs Petra Schütz for excellent technical assistance. The financial support by CEFIC is gratefully acknowledged.
Conflict of interest statement: None declared.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +49 731 500 65440; Fax: +49 731 500 65402; Email: guenter.speit{at}uni-ulm.de
|
References |
|---|
|
TopIntroductionMaterials and methodsResults and discussionFundingReferences |
|---|
-
1. IARC. Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol. IARC Monogr. Eval. Carcinog. Risks Hum. (2006) 88:1–478.[Medline]
2. Obe G, Beek B. Mutagenic activity of aldehydes. Drug Alcohol Depend. (1979) 4:91–94.[CrossRef][Web of Science][Medline]
3. Kreiger RA, Garry VF. Formaldehyde-induced cytotoxicity and sister-chromatid exchanges in human lymphocyte cultures. Mutat. Res. (1983) 120:51–55.[CrossRef][Web of Science][Medline]
4. 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]
5. 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.
6. Speit G, Schutz P, Hogel J, Schmid O. Characterization of the genotoxic potential of formaldehyde in V79 cells. Mutagenesis (2007) 22:387–394.
7. Wilson DM III, Thompson LH. Molecular mechanisms of sister-chromatid exchange. Mutat. Res. (2007) 616:11–23.[Web of Science][Medline]
8. TOXNET. Hazardous Substances Data Bank, Formaldehyde. (2007) (http://toxnet.nlm.nih.gov, accessed 14 March 2008).
9. Speit G, Schmid O, Neuss S, Schutz P. Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells. Environ. Mol. Mutagen. (2008) (in press).
10. Heck H, Casanova M. The implausibility of leukemia induction by formaldehyde: a critical review of the biological evidence on distant-site toxicity. Regul. Toxicol. Pharmacol. (2004) 49:300–307.
11. Golden R, Pyatt D, Shields PG. Formaldehyde as a potential human leukemogen: an assessment of biological plausibility. Crit. Rev. Toxicol. (2006) 36:135–153.[CrossRef][Web of Science][Medline]
Received on March 20, 2008; revised on April 15, 2008; accepted on April 15, 2008.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||