Previous studies have begun to elucidate the DNA repair and tolerance pathways that mitigate the genotoxic events following formaldehyde exposure. Specifically, formaldehyde has been shown to elicit cellular responses involving NER and HR pathways to overcome DNA damage. One of the critical players in the NER pathway is the XPF/ERCC1 complex that makes an incision 5′ to the lesion, followed by strand displacement and XPG-mediated 3′ incision [31
]. Interestingly, both biochemical and cellular studies support an additional role for XPF/ERCC1 in ICL repair [32
], where it is proposed to work in conjunction with the HR pathway (independent of NER) to remove the ICL lesion. Consistent with previously published data [19
], our study shows an increased formaldehyde sensitivity of XPF and ERCC1 mutants compared to other NER mutants, suggesting that the role of the XPF/ERCC1 complex is more critical than the other NER components for processing formaldehyde-induced DNA damage. One plausible explanation for the hypersensitive phenotype of the XPF and ERCC1 mutants, relative to other NER mutants may be that these proteins are involved in repair of both formaldehyde-induced DPCs and non-DPC lesions (such as ICLs), while the function of classical NER players remain confined to DPCs only. Alternatively, it is possible that both DPC and ICL repair mechanisms share common steps for processing and removal of the lesions. However, our studies in yeast clearly demonstrate that formaldehyde and ICL tolerance pathways are distinct but may have some common steps for processing and removal of formaldehyde-induced lesions [21
To elucidate the role of XPF endonuclease in repair of formaldehyde-induced DPCs and examine similarities and/or differences between ICL- and DPC-induced repair mechanisms in mammalian cells, we studied the formation and repair of DSBs that may be formed during the processing of formaldehyde-induced DPCs. It has been previously demonstrated that formaldehyde induces DNA DSBs as measured by the appearance of γH2AX foci; however, the nature of these DSBs and their repair kinetics have not been well studied, particularly in an XPF-deficient background. Unlike IR-induced DNA DSBs that have a very short half-life of minutes [39
], formaldehyde-induced DSBs are significantly long-lived as evidenced by the slow rate at which γH2AX foci declined in AA8 cells (an overall 15% decrease in a 24 hr period). A previous study conducted by Niedernhofer et al (2004) showed that MMC-induced DSBs occur via an ERCC1-independent mechanism; however, the DSBs persisted in the absence of ERCC1 suggesting that ERCC1 is required for the repair of MMC-induced DSBs. It is worth noting that our data on formaldehyde-induced DPCs are comparable to MMC-induced DSBs that also exhibit very slow repair kinetics as shown in ERCC1-deficent cells [40
]. Thus, it is tempting to speculate that MMC and formaldehyde-induced DSBs share common structural intermediates but differ from IR-induced DSBs. Relative to wild-type cells, XPF-deficient cells exhibited a slightly higher frequency of spontaneous γH2AX foci presumably due to DSBs arising from unrepaired endogenous DNA lesions. It is shown herein that analogous to MMC-induced DSBs [40
], most of the formaldehyde-induced DSBs are initiated during the S phase of the cell cycle and can be prevented by inhibiting replication elongation. The significance of the S phase-specific formaldehyde-induced DSBs may be associated with either lesion recognition, or processing of lesions resulting in a DSB at stalled replication forks. Our finding that formaldehyde-induced DSBs are generated in an XPF-independent manner favors a model in which the initial DSB is catalyzed by another endonuclease. However, when XPF function is compromised, DNA DSBs persist for longer and failure to repair these breaks eventually results in cell death. Collectively, these data support a role for XPF in the late steps of formaldehyde-induced DSB repair.
Many of the earlier studies on the formaldehyde-induced DNA damage response focused on studying the immediate effects of this agent and did not assess the cellular alterations following post treatment. This study demonstrates that the cytotoxic and genotoxic effects of formaldehyde may be a consequence of secondary lesions generated during processing of the primary adducts (i.e., DPCs converted to single-strand or double-strand breaks). This is supported by the flow cytometry and cytogenetic results demonstrating that the cell cycle changes and chromosomal rearrangements were more pronounced during recovery following formaldehyde treatment. However, in the absence of repair/tolerance mechanisms, these adverse effects were exhibited immediately and more severely. Specifically, the appearance of chromosomal breaks and radials at high formaldehyde concentrations (≥200 µM) in the wild-type background were observed only during the recovery period, whereas these effects were evident in the XPF-deficient cells immediately after treatment, even at the lowest concentration of formaldehyde (50 µM). Collectively, these findings highlight the cellular and cytogenetic abnormalities that develop following formaldehyde exposure, and also suggest that humans with compromised repair function(s) are at increased risks of formaldehyde-induced genomic instability. In mammalian cells, similar patterns of cell cycle profiles, DSB formation and repair kinetics, and chromosomal abnormalities, suggest that there is a substantial overlap between the repair mechanisms triggered by ICL-inducing agents and formaldehyde.
In addition to chromosomal breaks and radial formation, formaldehyde treatment also resulted in polyploidy and aneuploidy in both wild-type and XPF-deficient cells. Polyploidy and aneuploidy are believed to be a consequence of unequal chromosomal segregation, and it has been suggested that the presence of multiple centrosomes can subsequently promote either bipolar or multipolar cell divisions, leading to chromosomal mis-segregation and abnormalities [28
]. However, viable progenies result mostly from bipolar cell divisions but not multipolar cell divisions [41
]. Our data suggest that formaldehyde-treated cells accumulate centrosome abnormalities and that following bipolar cell divisions, these may result in daughter cells containing mis-segregated chromosomes as evidenced by the ploidy changes demonstrated by flow cytometry and cytogenetic analyses.