Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental human carcinogen. Whereas humans are continuously exposed to exogenous formaldehyde, this reactive aldehyde is a naturally occurring biological compound that is present in human plasma at concentrations ranging from 13 to 97 μmol/L. It has been well documented that DNA-protein crosslinks (DPC) likely play an important role with regard to the genotoxicity and carcinogenicity of formaldehyde. However, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde. In the present study, we first assessed the DNA damage response to plasma levels of formaldehyde using chicken DT40 cells with targeted mutations in various DNA repair genes. Here, we show that the hypersensitivity to formaldehyde is detected in DT40 mutants deficient in the BRCA/FANC pathway, homologous recombination, or translesion DNA synthesis. In addition, FANCD2-deficient DT40 cells are hypersensitive to acetaldehyde, but not to acrolein, crotonaldehyde, glyoxal, and methylglyoxal. Human cells deficient in FANCC and FANCG are also hypersensitive to plasma levels of formaldehyde. These results indicate that the BRCA/FANC pathway is essential to counteract DPCs caused by aliphatic monoaldehydes. Based on the results obtained in the present study, we are currently proposing that endogenous formaldehyde might have an effect on highly proliferating cells, such as bone marrow cells, as well as an etiology of cancer in Fanconi anemia patients. [Cancer Res 2007;67(23):11117–22]
- DNA-protein crosslink
- Fanconi anemia
Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental pollutant found in automobile emissions and tobacco smoke. Recently, formaldehyde has been reevaluated by the IARC as a known environmental human carcinogen (group 1) due to a positive association between the extent of formaldehyde exposure of workers and their death from nasopharyngeal cancer ( 1). Whereas humans are continuously exposed to exogenous formaldehyde, this chemical is also a naturally occurring biological compound that is present in all tissues, cells, and bodily fluids. Formaldehyde and its oxidation product, formic acid, function as key intermediates in the “one-carbon pool” ( 2) used for the biosynthesis of purines, thymidine, and some amino acids. It has been found that the concentration of endogenous formaldehyde in human plasma ranges from 13 to 97 μmol/L ( 3, 4), with Heck et al. reporting an average of 82 μmol/L (calculated using 1.060 for the density of human blood). Formaldehyde is usually rapidly metabolized by reduction, oxidation, and reduced glutathione (GSH)–dependent pathways. However, saturation in formaldehyde metabolism may lead to DNA damage. It has been well documented that cells exposed to formaldehyde exhibit, as a major form of DNA damage, DNA-protein crosslinks (DPC; ref. 5). DNA-histone crosslinks have been shown to have a strong correlation with formaldehyde-induced tumor incidence in animals. As a result, the level of formaldehyde-induced DPCs is considered to be a good molecular dosimeter for formaldehyde cancer risk assessment ( 5). Although understanding the importance of DPCs with regard to the genotoxicity of formaldehyde is paramount, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde.
Fanconi anemia is characterized by developmental abnormalities, susceptibility to certain cancers, and sensitivity to DNA-DNA crosslinking agents ( 6). At least 13 FANC complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, M, N) have been identified ( 6). These proteins interact in a common pathway that activates FANCD2 via monoubiquitination ( 6). Upon recognition of a stalled replication fork, for instance, by the nuclear E3 monoubiquitin ligase core complex, activated FANCD2 is targeted to BRCA1 nuclear foci where it regulates DNA repair by possibly homologous recombination and translesion synthesis ( 6). DT40 cells and their isogenic mutants have predominantly been used to investigate the function of various gene products ( 7). In the present study, we assessed the DNA damage response to formaldehyde by the reverse genetic approach using the DT40 cell model system and isogenic human cancer cells deficient in the FANC pathway.
Materials and Methods
Materials. Fetal bovine serum (FBS), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT), 1-methoxy-5-methylphenazinium methyl sulfate, acetaldehyde, acrolein, glyoxal, and methylglyoxal were obtained from Sigma. Formaldehyde and crotonaldehyde were purchased from Fisher Scientific and Across Organics, respectively. The total GSH quantification kit was from Dojindo Molecular Technologies, Inc. RPMI 1640 culture medium, chicken serum, and penicillin/streptomycin were obtained from Invitrogen.
Cell lines and cell culture. All DT40 mutants were derived from isogenic DT40 parent cell lines (Supplementary Table S1). The DT40 cells and their mutants were cultured as previously reported ( 8). Parental colorectal cancer cell lines (RKO) cells and isogenic clones harboring an engineered disruption of FANCC or FANCG ( 9) were cultured in a humidified 5% CO2 atmosphere at 37°C. The medium consisted of RPMI 1640 cell culture medium containing 10% FBS (heat inactivated), 100 mg/mL penicillin, and 100 mg/mL streptomycin.
Cell survival assay. For DT40 cells and their mutants, suspended cells (∼600 cells per 250 μL per well) were seeded into 24-well plates, exposed to formaldehyde (and other aldehydes), and allowed to divide for ∼10 cycles. The formaldehyde used was 37% aqueous and was serially diluted in sterile 1× PBS (Life Technologies) to obtain the appropriate concentrations in the plates. All formaldehyde dilutions were made fresh and kept on ice. (Initially, a colony formation assay was attempted, but discontinued, as it seemed formaldehyde reacted adversely with the methylcellulose semisolid medium). After cultivation, cell viability was determined by the XTT assay ( 10). For RKO cells and their isogenic cells deficient in FANCC or FANCG, the adhesive cells (∼600 cells per 250 μL per well) were seeded into 24-well plates and cultivated for 2 days before treatment. After changing to fresh medium, the cells were exposed to formaldehyde and allowed to divide for 7 days. The medium was replenished at 4 days after treatment. The survival rates were determined as described above.
Determination of intracellular total GSH. Total GSH levels were measured according to the manufacturer's directions using a commercially available kit.
Statistical analysis. Survival data were log-transformed giving approximate normality. Analysis of covariance (ANCOVA) was used to test for mean intercept differences and differences in the slopes of the linear dose-response curves between wild-type and a series of mutant cells.
Results and Discussion
Homologous recombination repair and nonhomologous end joining repair of DNA damage induced by formaldehyde. We assessed the contribution of each DNA damage response pathway to formaldehyde-induced DNA damage. Toward this end, we used a panel of isogenic DT40 cell mutants (Supplementary Table S1), each of which was defective in a particular DNA repair or cell cycle checkpoint response pathway. Here, we show that formaldehyde causes a reduction in survival of DT40-derived mutants that are deficient in homologous recombination repair. The homologous recombination repair pathway mutants showed sensitivity to formaldehyde in the following order: FANCD2 >> BRCA2 > BRCA1 = XRCC2 = RAD51C = RAD51D = XRCC3 = RAD54 > RAD52 > parent DT40 cells ( Fig. 1A and B ; Supplementary Fig. S1A and B). In contrast, cell survival rates were largely equivalent between nonhomologous end joining (NHEJ)–deficient cells and parent DT40 cells ( Fig. 1A and B; Supplementary Fig. S1C). One of the metabolic pathways used by cells to detoxify formaldehyde is GSH-dependent. Therefore, we tested whether there is an association between intracellular GSH levels and hypersensitivity of homologous recombination–deficient cells to formaldehyde. However, we found no correlation between GSH concentrations and formaldehyde-induced cell toxicity in series of isogenic DT40 cells (data not shown). These results revealed a requirement for the homologous recombination pathway, but not the NHEJ pathway, in processing DNA damage induced by formaldehyde, strongly suggesting that the homologous recombination pathway is involved in repair of DPCs.
Role of excision repair pathways in the repair of DNA damage induced by formaldehyde. Results of biochemical experiments have indicated that nucleotide excision repair (NER) is involved in the elimination of DPCs ( 11, 12). However, the relative contribution of the various excision repair pathways to the removal of DPCs has not been fully characterized. Therefore, we tested each excision repair pathway [NER and base excision repair (BER)] as to its effectiveness in removing DPCs caused by formaldehyde. Our results show hypersensitivity in the following order: PARP1 (BER/single-strand and double-strand break repairs) > XPA (NER) > POLβ (BER) > FEN1 (BER; Fig. 1A and B; Supplementary Fig. S1D). The DT40 mutants for POLβ and FEN1 showed slight to marginal hypersensitivity, indicating that BER is not heavily involved in DPC repair. PARP1 is used in many pathways, such as BER, single-strand break repair, stabilization of replication forks, and possibly double-strand break repair ( 13). Thus, because POLβ and FEN1 sensitivities indicate that BER is probably not highly implicated in formaldehyde-induced DPC repair, the higher sensitivity of the PARP1 mutant may be due to its role in DNA replication and double-strand break repair in cells exposed to formaldehyde. In addition to PARP1, XPA likely plays a moderate role for counteracting DNA damage induced by formaldehyde. These results obtained from XPA-deficient DT40 cells agree with a previous study which showed that human fibroblasts deficient in NER (XPA and XPF) were slightly hypersensitive to formaldehyde in cell survival ( 14).
Role of translesion DNA synthesis and cell cycle checkpoint pathways in the repair of DNA damage induced by formaldehyde. In the DT40 cell model system, REV1-deficient, REV3-deficient, and RAD18-deficient cells have been reported to be hypersensitive to various DNA-DNA intercrosslinking agents, including cisplatin and mitomycin C ( 15). To determine the role of translesion synthesis in the repair of DNA damage induced by formaldehyde, we exposed DT40 and DT40 cells deficient in REV1, REV3, and RAD18 along with POL𝛉 to formaldehyde. For the REV1, REV3, and RAD18 mutants, our results show good agreement with the intercrosslinking study, whereas POL𝛉 mutants were only marginally sensitive to formaldehyde ( Fig. 1A and B; Supplementary Fig. S1E). Proliferating cell nuclear antigen (PCNA) has been reported to be partly ubiquitinated in a RAD18-dependent manner, and this monoubiquitination of PCNA is necessary for carrying out translesion synthesis by polymerase η in yeasts ( 16). In addition, it has been proposed that ATR/CHK1 signaling is required for RAD18-mediated PCNA monoubiquitination against DNA damage induced by benzo(a)pyrene dihydrodiol epoxide ( 17). To further characterize the DNA damage response to formaldehyde, we exposed formaldehyde to cells deficient in the S-phase cell cycle components ATM or CHK1. Formaldehyde sensitivity in these cells was similar to parental cells ( Fig. 1A and B; Supplementary Fig. S1F). These results suggest that neither the ATM nor CHK1 pathway participate in the DNA damage response to formaldehyde-induced DNA damage.
Role of FANCD2 in the repair of DNA damage induced by endogenously existing aldehydes other than formaldehyde. Using DT40 cells deficient in FANCD2, the most sensitive cell line to formaldehyde, we addressed whether other major endogenously existing aldehydes cause similar hypersensitivity to FANCD2 cells. Among the endogenously existing aldehydes potentially capable of inducing DPCs ( 18), acetaldehyde caused hypersensitivity in FANCD2 cells at millimolar levels ( Fig. 2A ). Methylglyoxal ( Fig. 2B), glyoxal (data not shown), acrolein, and crotonaldehyde ( Fig. 2C and D) showed no hypersensitivity in FANCD2 cells ( Fig. 2B–D). These results suggest that the FANC pathway may be essential to counteract DPCs induced by aliphatic short monoaldehydes, but not DPCs caused by either aliphatic dicarbonyl compounds or α,β-unsaturated aldehydes. Another possibility is that either α,β-unsaturated aldehydes or aliphatic dicarbonyl compounds may more efficiently introduce other types of deleterious DNA lesions, such as exocyclic base adducts or oxidative base lesions.
Endogenous DPC-inducing agents and Fanconi anemia. Fanconi anemia, a rare disease with heterozygosity existing at a frequency of ∼0.4% of the population ( 19), is an inherited disorder associated with progressive bone marrow failure and predisposition to malignant leukemia and solid tumors ( 6). It has been well documented that cells from Fanconi anemia patients are hypersensitive to DNA interstrand crosslinking agents ( 6), with an associated increase in chromosomal breakage. We know, however, of no report that has described a condition whereby endogenous reactive agents, such as formaldehyde, have induced DPCs with the further induction of toxicity in cells deficient in the FANC pathway. It has been reported that endogenous formaldehyde in human plasma is detectable at concentrations ranging from 13 to 97 μmol/L ( 3, 4). The two most sensitive DT40 mutants are FANCD1 (BRCA2)–deficient and FANCD2-deficient cells, which showed hypersensitivity to formaldehyde at concentrations between 10 and 15 μmol/L. This observation raises a question about the relevance of data derived from the DT40 model system to the human cellular response to formaldehyde. Therefore, we exposed RKO cells and their isogenic cells disrupted in FANCC or FANCG. The human cells deficient in either FANCC or FANCG were hypersensitive to formaldehyde at concentrations 20 μmol/L or higher (FANCC) or 38 μmol/L or higher (FANCG; Fig. 3 ). These results indicate that the FANC/BRCA pathway plays a critical function in not only DT40 cells, but in human cells. Therefore, we currently hypothesize that endogenous formaldehyde induces DPCs and plays a critical role in the initiation of progressive bone marrow failure or predisposition to malignant tumors in Fanconi anemia patients.
Possible DNA repair pathways for DPCs induced by formaldehyde. It is widely accepted that formaldehyde predominantly introduces DPCs in cells. The DPC formation is believed to be related to formaldehyde-induced cancer in animals and humans; however, little information is available in terms of the DNA damage response to formaldehyde. Interestingly, we recognized that the DNA damage responses to formaldehyde in the DT40 cell model system were similar to those of cisplatin ( 15). For example, survival of isogenic DT40 mutants to cisplatin is in the following order: REV3 > FANCC > RAD18 > XRCC2 > XRCC3 > PARP1 > RAD54 > BRCA1 > XPA = ATM > BRCA2 = MSH3 > parental DT40 cells. Because both DNA-DNA crosslinks and DPCs have the potential to cause stalled DNA replication forks and may introduce double-strand breaks, hypersensitivity of homologous recombination–deficient cells to formaldehyde is likely due to DPCs. Furthermore, cisplatin induces DPCs in addition to interstrand or intrastrand crosslinks. The similarity of the DNA damage response in DT40 mutants to cisplatin and formaldehyde also raised the possibility that DPCs caused by cisplatin may have a role in the hypersensitivity of DT40 cells deficient in the FANC/BRCA and homologous recombination pathways. We also found hypersensitivity of PARP1-deficient DT40 cells to formaldehyde. Interestingly, PARP1 also inhibits homologous recombination from interference by Ku and ligase IV in DT40 cells ( 20). This report, combined with our results, suggests that without precise regulation of homologous recombination in the absence of PARP1, the homologous recombination repair pathway for the repair of double-strand breaks induced by DPCs may not efficiently restore the integrity of genomic DNA. Although XPA mutants are more resistant to formaldehyde compared with homologous recombination–deficient cells (a characteristic shared with cisplatin; ref. 15), XPA-deficient cells were moderately hypersensitive to formaldehyde, suggesting a reasonable role for NER in the elimination of DNA damage caused by formaldehyde. Figure 4 shows a possible DNA repair pathway to counteract DPCs induced by formaldehyde. In the error-free DPC excision process, formaldehyde-induced DPCs could be first degraded to DNA–amino acid crosslinks (DAC) by a cellular proteasome. The NER pathway may recognize and eliminate the DACs followed by DNA repair synthesis ( 11, 12, 14). In contrast, error-prone DPC excision processes could exist to tolerate formaldehyde-induced damage. After degradation of DPCs to DACs but before initiation of excision repair, DNA replication may start and translesion synthesis DNA polymerases may by-pass and extend DNA synthesis past the DACs. After DNA replication, the NER pathway may recognize and eliminate the DACs followed by DNA repair synthesis. Because homologous recombination–deficient cells are hypersensitive to formaldehyde, the requirement for homologous recombination repair for uncoupling of initiation of DNA replication and excision repair processes can be hypothesized. The NER pathway recognizes and eliminates DACs. Before the completion of excision repair, DNA replication could begin and lead to the formation of DNA double-strand breaks with subsequent repair by a homologous recombination–dependent pathway. Another possibility is that DPCs formed during DNA replication cause stalled DNA replication forks, followed by the formation of DNA double-strand breaks. The homologous recombination–dependent pathway could then repair these DNA double-strand breaks.
Grant support: Center for Environmental Health and Susceptibility grant NIEHS P30-ES10126, Superfund Basic Research Program grant NIEHS P42-ES05948, and University of North Carolina at Chapel Hill Department of Environmental Sciences and Engineering B.B. Parker Fellows Program, and Public Health Service training grant (J.R. Ridpath).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Tadayoshi Bessho, Dr. Elizabeth Fryar Tita, Dr. Nadia Georgieva, Dr. Scott Blutman, and Brian F. Pachkowski for critically reading the manuscript and Dr. Scott E. Kern for providing isogenic RKO cells.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Current address for M. Takata: Radiation Biology Center, Kyoto University, Kyoto, Japan, and M. Yamazoe: Wakayama Medical University, School of Medicine, Wakayama, Japan.
- Received August 7, 2007.
- Revision received September 24, 2007.
- Accepted October 11, 2007.
- ©2007 American Association for Cancer Research.