Formaldehyde-induced DNA Damage Research Articles

Reducing DNA Damage Associated with Endogenous Genotoxic Stress in Hematopoietic Stem Cells

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. Endogenous formaldehyde is ubiquitous within cells as it is produced as a byproduct during normal cellular metabolism such as histone, RNA and DNA demethylation. Previous studies show that endogenous formaldehyde is a hematopoietic stem cell (HSC) genotoxin and metabolic carcinogen. We previously showed that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5), and Adh5-deficient mice therefore accumulate formaldehyde adducts in DNA that require the FA pathway to repair. Mice lacking Adh5 together with the FA pathway central component Fancd2 exhibit bone marrow failure and early lethality. Adh5 haploinsufficiency combined with Fancd2 deficiency, or Fancd2 -/- Adh5 +/- mice, demonstrates severely compromised HSC activity. However, the contributing factors that cause DNA damage and the protective mechanisms by which cells reduce DNA damage during endogenous genotoxic stress are not fully understood. Here, we show that the genetic inactivation of Lnk/Sh2b3 in Fancd2 -/- Adh5 +/- mice restores HSC function. Adh5 haploinsufficiency exacerbated Fancd2 deficiency, while Lnk deficiency rescued HSC homeostasis and transplantability of Fancd2 -/- Adh5 +/- mice. Moreover, Lnk deficiency increased HSC quiescence and reduced endogenous DNA damage in Fancd2 -/- Adh5 +/- mice. Importantly, Lnk deficiency does not participate in repairing formaldehyde-induced DNA damage, nor does it metabolize formaldehyde or reduce genomic formaldehyde-deoxyguanine adduct in vivo. Rather, Lnk deficiency reduced single-strand DNA (ssDNA) breaks and ssDNA binding protein RPA in HSCs during the cell cycle, thereby suppressing the ATR pathway activation upon endogenous replication stress. Moreover, loss of Lnk increased S-phase progression and fork recovery in Fancd2 -/- Adh5 +/- HSPCs at the stressed replication fork upon HU treatment, thereby preventing replication fork collapse and DNA damage. These findings highlight a new mechanism by which Lnk deficiency mitigates replication stress-induced DNA damage in response to endogenous genotoxic insult from cellular metabolism during HSC regeneration.

Digenic mutations in ALDH2 and ADH5 impair formaldehyde clearance and cause a multisystem disorder, AMeD syndrome.

Rs671 in the aldehyde dehydrogenase 2 gene (ALDH2) is the cause of Asian alcohol flushing response after drinking. ALDH2 detoxifies endogenous aldehydes, which are the major source of DNA damage repaired by the Fanconi anemia pathway. Here, we show that the rs671 defective allele in combination with mutations in the alcohol dehydrogenase 5 gene, which encodes formaldehyde dehydrogenase (ADH5FDH ), causes a previously unidentified disorder, AMeD (aplastic anemia, mental retardation, and dwarfism) syndrome. Cellular studies revealed that a decrease in the formaldehyde tolerance underlies a loss of differentiation and proliferation capacity of hematopoietic stem cells. Moreover, Adh5-/-Aldh2 E506K/E506K double-deficient mice recapitulated key clinical features of AMeDS, showing short life span, dwarfism, and hematopoietic failure. Collectively, our results suggest that the combined deficiency of formaldehyde clearance mechanisms leads to the complex clinical features due to overload of formaldehyde-induced DNA damage, thereby saturation of DNA repair processes.

The multiplex interactions and molecular mechanism on genotoxicity induced by formaldehyde and acrolein mixtures on human bronchial epithelial BEAS-2B cells

Aldehydes are common air pollutants with carcinogenicity. Genotoxicity of single aldehyde has been studied well, but the combined genotoxicity is rarely known. Here, we evaluated the combined genotoxicity of formaldehyde and acrolein on BEAS-2B cells in terms of DNA strands breakage, chromosome damage and gene mutation below subcytotoxic concentrations covering smoking-related concentrations. Meanwhile, the molecular mechanism was investigated further based on oxidative stress, DNA-protein crosslinks (DPCs), cell cycle and DNA damage-repair pathway. Co-exposure to formaldehyde and acrolein mixtures showed significantly synergistic interaction on DNA strands breakage and chromosome damage in a concentration/time-dependent manner, while antagonism was shown on the late genotoxic endpoints (e.g. cytoplasmic block micronucleus (CBMN) and HPRT gene mutation). Moreover, formaldehyde synergistically potentiated acrolein-induced S-phase arrest, inhibition of DNA repair and up-regulation of genes related to cell stress, which conversely strengtherned mixture-induced DNA/chromosome damage and finally resulted in antagonism on late genotoxic events. Additionally, formaldehyde-induced DNA damage mainly resulted from the direct covalent bonding (e.g. DPCs), while acrolein-induced DNA damage mainly generated from oxidative damage (e.g. oxidative stress), which dominated the synergistic DNA strand breakage induced by mixtures. Summarily, aldehyde mixtures (formaldehyde and acrolein) induced multiplex combined genotoxicity on BEAS-2B cells even at smoking-related concentrations, which was dependent on genotoxic endpoints and closely related to that formaldehyde potentiated acrolein-induced cell stress, S-phase arrest and inhibition of DNA repair. So prolonged exposure to aldehyde mixtures may have a more serious risk to respiratory system in animal and human than the expectation based on the toxicity of single aldehyde even at environmentally relevant concentrations.

Genetic investigation of formaldehyde-induced DNA damage response in Schizosaccharomyces pombe

Formaldehyde is a common environmental pollutant and is associated with adverse health effects. Formaldehyde is also considered to be a carcinogen because it can form DNA adducts, leading to genomic instability. How these adducts are prevented and removed is not fully understood. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism to investigate cellular tolerance pathways against formaldehyde exposure. We show that Fmd1 is a major formaldehyde dehydrogenase that functions to detoxify formaldehyde and that Fmd1 is critical to minimize formaldehyde-mediated DNA lesions. Our investigation revealed that nucleotide excision repair and homologous recombination have major roles in cellular tolerance to formaldehyde, while mutations in the Fanconi anemia, translesion synthesis, and base excision repair pathways also render cells sensitive to formaldehyde. We also demonstrate that loss of Wss1 or Wss2, proteases involved in the removal of DNA-protein crosslinks, sensitizes cells to formaldehyde and leads to replication defects. These results suggest that formaldehyde generates a variety of DNA lesions, including interstrand crosslinks, DNA-protein crosslinks, and base adducts. Thus, our genetic studies provide a framework for future investigation regarding health effects resulting from formaldehyde exposure.

Evaluation of Inhaled Low Dose Formaldehyde Induced DNA Damage by Liquid Chromatography-Tandem Mass Spectrometry

Evaluation of Inhaled Low Dose Formaldehyde Induced DNA Damage by Liquid Chromatography-Tandem Mass Spectrometry

Endogenous Formaldehyde Is a Hematopoietic Stem Cell Genotoxin and Metabolic Carcinogen

SummaryEndogenous formaldehyde is produced by numerous biochemical pathways fundamental to life, and it can crosslink both DNA and proteins. However, the consequences of its accumulation are unclear. Here we show that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), and Adh5−/− mice therefore accumulate formaldehyde adducts in DNA. The repair of this damage is mediated by FANCD2, a DNA crosslink repair protein. Adh5−/−Fancd2−/− mice reveal an essential requirement for these protection mechanisms in hematopoietic stem cells (HSCs), leading to their depletion and precipitating bone marrow failure. More widespread formaldehyde-induced DNA damage also causes karyomegaly and dysfunction of hepatocytes and nephrons. Bone marrow transplantation not only rescued hematopoiesis but, surprisingly, also preserved nephron function. Nevertheless, all of these animals eventually developed fatal malignancies. Formaldehyde is therefore an important source of endogenous DNA damage that is counteracted in mammals by a conserved protection mechanism.

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage.

Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N(2-)hydroxymethyl-dG (N(2)-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N(2)-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [(13)CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N(2)-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair.

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

Role of PARP-1 in formaldehyde-induced DNA damage repair and apoptosis in human bronchial epithelial cells

To study the role of poly (ADP-ribose) polymerase-l (PARP-1) in formaldehyde-induced DNA damage response in human bronchial epithelial (HBE) cells and to investigate the mechanism of formaldehyde carcinogenicity. The protein levels were measured by Western blot. The interaction between different proteins was determined by co-immunoprecipitation assay. The chemical inhibitor was used to confirm the relationship between PARP-1 and DNA damage repair. After being exposed to different concentrations of formaldehyde for 4 h, HBE cells showed no significant changes in cell viability. Cell viability was significantly reduced after 24-h exposure to 80 and 160 µmol/L formaldehyde (P < 0.05). The 10 µmol/L formaldehyde resulted in significant increases in the protein levels of PARP-1 and XRCC-1. However, 80 µmol/L formaldehyde led to a significant decrease in the protein level of PARP-1 of 124 KD molecular weight but a significant increase in the protein level of PARP-1 of 89 KD molecular weight; there was no significant change in the protein level of XRCC-1. The co-immunoprecipitation assay showed that 10 µmol/L formaldehyde induced increased binding between PARP-1 and XRCC-1, but 80 µmol/L formaldehyde led to no significant change in binding between PARP-1 and XRCC-1. Here, we confirmed the role of 10 µmol/L formaldehyde in strand breaks by comet assay which showed an increase in the tail DNA content of HBE cells after 4-h formaldehyde exposure. No significant difference was observed in tail DNA content between treated HBE cells and control cells at 2 h after formaldehyde was removed. Moreover, compared with control, inhibition of PARP-1 induced a significant increase in tail DNA content, and a significant difference was observed in tail DNA content between inhibited HBE cells and control cells at 2 h after formaldehyde was removed. Inhibition of PARP-1 significantly reduced DNA repair capacity. PARP-1 mediated the repair of DNA damage induced by low-concentration formaldehyde through recruiting XRCC-1 protein, and may be involved in the regulation of cell apoptosis induced by high-concentration formaldehyde.

Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway

Formaldehyde is a reactive chemical that is commonly used in the production of industrial, laboratory, household, and cosmetic products. The causal association between formaldehyde exposure and increased incidence of cancer led the International Agency for Research on Cancer to classify formaldehyde as a carcinogen. Formaldehyde-induced DNA–protein crosslinks (DPCs) elicit responses involving nucleotide excision repair (NER) and homologous recombination (HR) repair pathways; however, little is known about the cellular and genetic changes that subsequently lead to formaldehyde-induced genotoxic and cytotoxic effects. Herein, investigations of genes that modulate the cytotoxic effects of formaldehyde exposure revealed that of five NER-deficient Chinese Hamster Ovary (CHO) cell lines tested, XPF- and ERCC1-deficient cells were most sensitive to formaldehyde treatment as compared to wild-type cells. Cell cycle analyses revealed that formaldehyde-treated XPF-deficient cells exhibited an immediate G2/M arrest that was associated with altered cell ploidy and apoptosis. Additionally, an elevated number of DNA double-strand breaks (DSBs), chromosomal breaks and radial formation were also observed in XPF-deficient cells following formaldehyde treatment. Formaldehyde-induced DSBs occurred in a replication-dependent, but an XPF-independent manner. However, delayed DSB repair was observed in the absence of XPF function. Collectively, our findings highlight the role of an XPF-dependent pathway in mitigating the sensitivity to formaldehyde-induced DNA damage as evidenced by the increased genomic instability and reduced cell viability in an XPF-deficient background. In addition, centrosome and microtubule abnormalities, as well as enlarged nuclei, caused by formaldehyde exposure are demonstrated in a repair-proficient cell line.

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA−/−, FANCC−/−, FANCA−/−C−/−, FANCD2−/− and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA−/−, FANCC−/− and FANCA−/−C−/− cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2−/− cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow

Arsenic is a human skin, lung, and urinary bladder carcinogen, and may act as a cocarcinogen in the skin and urinary bladder. Possible modes of action of arsenic carcinogenesis/cocarcinogenesis include oxidative stress induction and inhibition of DNA damage repair. We investigated the effects of arsenic in drinking water on DNA damage repair in urinary bladder transitional cells and on micronucleus formation in bone marrow. F344 rats were given 100 ppm arsenate [As(V)] or dimethylarsinic acid [DMA(V)] in drinking water for 1 week. The in vivo repair of cyclophosphamide (CP)-induced DNA damage resulting from a single oral gavage of CP, and the in vitro repair of hydrogen peroxide (H(2)O(2))- or formaldehyde-induced DNA damage, resulting from adding H(2)O(2) or formaldehyde into cell medium, were measured by the Comet assay. DMA(V) effects were not observed on either CP-induced DNA damage induction or on DNA repair. Neither DMA(V) nor As(V) increased the H(2)O(2)- or formaldehyde-induced DNA damage, and neither inhibited the repair of H(2)O(2)-induced DNA damage. Neither DMA(V) nor As(V) increased the micronucleus frequency, nor did they elevate micronucleus frequency resulting from CP treatment above the level observed by the treatment with CP alone. These results suggest that arsenic carcinogenesis/cocarcinogenesis in the urinary bladder may not be via DNA damage repair inhibition. To our knowledge this is the first report of arsenic effects on DNA damage repair in the urinary bladder.

Benzo[a]pyrene- and formaldehyde-induced DNA damage and repair in rat tracheal epithelial cells

The single and combined effects of 2 environmental carcinogens, benzo[a]pyrene (BAP) and formaldehyde (HCHO), on cell growth and DNA damage were assessed in a rat tracheal epithelial cell line, C18. Treatment of C18 cells with HCHO for 90 min reduced the calculated growth index at the highest concentration tested, 400 μM, while no growth effects were observed with BAP treatments. Combination treatments reduced the growth index to 75% of control values. Alkaline elution analysis of C18 cell DNA detected both DNA-protein crosslinks (DPC) and single-strand breaks (SSB) as a result of HCHO treatment, while BAP caused only SSB. HCHO-induced SSB were repaired within 2 h, whereas BAP-induced SSB persisted for at least 48 h. Combination treatment of cells with BAP followed by HCHO enhanced the number of SSB, but reduced DPC. The results are discussed in reference to earlier work which demonstrated the interaction in vivo between BAP and HCHO with respect to their carcinogenicity in rat tracheal implants.