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. 2015 Oct 1;60(1):177-88.
doi: 10.1016/j.molcel.2015.08.020. Epub 2015 Sep 24.

Endogenous Formaldehyde Is a Hematopoietic Stem Cell Genotoxin and Metabolic Carcinogen

Lucas B Pontel  1 , Ivan V Rosado  2 , Guillermo Burgos-Barragan  1 , Juan I Garaycoechea  1 , Rui Yu  3 , Mark J Arends  4 , Gayathri Chandrasekaran  5 , Verena Broecker  6 , Wei Wei  7 , Limin Liu  7 , James A Swenberg  3 , Gerry P Crossan  1 , Ketan J Patel  8
Affiliations

Endogenous Formaldehyde Is a Hematopoietic Stem Cell Genotoxin and Metabolic Carcinogen

Lucas B Pontel et al. Mol Cell. .

Abstract

Endogenous 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.

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Figures

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Graphical abstract
Figure 1
Figure 1
ADH5 Prevents the Accumulation of Endogenous Formaldehyde DNA Adducts (A) Scheme outlining the origin and catabolism of endogenous formaldehyde by ADH5. MPO, myeloperoxidase; ADH, alcohol dehydrogenase. (B) Upper panel, immunoblot of whole cell extracts from wild-type (WT) and Adh5−/− mouse tissues probed with affinity-purified rabbit anti-ADH5 antiserum. β-actin was used as loading control. Lower panel shows an immunoblot of total kidney extract from wild-type mice loaded as 2-fold dilution series, comparing the relative expression of ADH5 with bone marrow. (C) Formaldehyde reacts with guanine to form the N2-hydroxymethyl-dG adduct, which can then be detected and quantified by mass spectrometry after reduction to N2-methyl-dG. (D) Bar chart representing the frequency of N2-methyl-dG per 107 dG bases in genomic DNA, obtained from bone marrow, kidney, and liver of WT or Adh5−/− mice at 10–15 weeks or following 4 weeks of methanol exposure. ∗∗p < 0.01; ∗p < 0.05. Data are represented as mean ± SEM.
Figure 2
Figure 2
Combined Genetic Inactivation of Adh5 and Fancd2 Leads to Rapid Loss of HSCs (A) Kaplan-Meier curve of the survival of Adh5−/−Fancd2−/− mice compared to allelic controls. (B) Quantification of red blood cells in peripheral blood of 4- to 6-week-old Adh5−/−Fancd2−/− mice and age-matched controls; each point represents a single mouse. ∗∗∗∗p < 0.0001. (C) Quantification of nucleated bone marrow cellularity in Adh5−/−Fancd2−/− mice and controls. ∗∗∗∗p < 0.0001; n = 14 per control group, and n = 22 in Adh5−/−Fancd2−/− group. (D) Left, representative flow cytometry plots showing 50,000 lineage− cells, used to quantify the HSPC pool in wild-type, Adh5−/−, Fancd2−/−, and Adh5−/−Fancd2−/− bone marrow (as LKS: Lin−c-kit+Sca-1+). Right, HSC frequency was quantified in the bone marrow of age-matched mice using LKS markers or in combination with alternative cell surface markers (SLAM: CD41−CD48−CD150+). Bar graphs show the mean relative to wild-type. n = 4 per group; ∗∗p < 0.01; ∗p < 0.05. (E) Frequency of CFU-S10 assessed following injection of 1 × 105 (control mice) or 2 × 106 (Adh5−/−Fancd2−/− mice) nucleated bone marrow cells into irradiated recipients. Each point represents the number of spleen colonies (CFU-S10) per recipient. ∗∗p < 0.01; n = 10 and 8 per control and Adh5−/−Fancd2−/− groups, respectively. (F–G) The long-term competitive repopulation assay was performed by transplanting 0.2 × 106 (F) or 5 × 106 (G) “test” cells from wild-type, Adh5−/−, Fancd2−/−, or Adh5−/−Fancd2−/− mice (CD45.2) together with 0.2 × 106 wild-type competitor cells (CD45.1) into irradiated recipients (CD45.1/CD45.2). The plots show the test/competitor chimerism in peripheral white blood cells over time. Data are represented as mean ± SEM. See also Figures S1 and S2.
Figure 3
Figure 3
ADH5 and FANCD2 Suppress DNA Damage in Hematopoietic Cells (A) Immunoblot showing the expression of ADH5 in different hematopoietic populations isolated from the bone marrow of 10-week-old wild-type mice by flow cytometry. The total protein fraction was isolated from 100,000 cells, and histone H3 was used as loading control. (B) Flow cytometric analysis of γ-H2A.X induction within the lineage negative (Lin−), Lin−c-kit+Sca-1− (LK) and Lin−c-kit+Sca-1+ (LKS) populations in the bone marrow obtained from Adh5−/−Fancd2−/− and control mice. The bar graph shows the γ-H2A.X fluorescence intensity relative to the wild-type control. n = 4; ∗p < 0.05. (C) Flow cytometry detection of γ-H2A.X induction in the LKS population (HSPC). (D) Metaphase spreads were prepared from LPS-activated splenic B cells and scored blinded for the presence of chromosome aberrations (wild-type n = 85, Adh5−/− n = 93, Fancd2−/− n = 91, and Adh5−/−Fancd2−/− n = 95 metaphases). Representative images of Adh5−/−Fancd2−/− metaphases are shown on the right (with a chromatid break and a radial structure indicated by black arrows). Scale bar, 10 μm. Data are represented as mean ± SEM. See also Figure S3.
Figure 4
Figure 4
Formaldehyde Drives HSCs Attrition in Adh5−/−Fancd2−/− Mice (A) Graph showing the survival of splenic B cells following exposure to formaldehyde. B cells were activated with LPS and were grown in the presence of formaldehyde for 6 days, and the viable cell number was assessed by trypan blue exclusion. (B) Plot showing the sensitivity of erythroid colony-forming units (CFU-E) to formaldehyde. Bone marrow-derived cells (2 × 106) were exposed for 2 hr to varying doses of formaldehyde and plated onto methylcellulose medium. In both (A) and (B), the survival was made relative to the untreated sample. The mean of three independent experiments is shown, each carried out in duplicate. ∗p < 0.05; ∗∗p < 0.01. (C) Top, scheme outlining the protocol used to assess the toxicity of methanol to HSCs. Six-week-old mice were fed with methanol 15% v/v in the water supply, and HSC frequency was quantified after 4 weeks. Bottom, the graph shows the flow cytometric quantification of HSCs (SLAM-LKS markers) for Adh5+/−Fancd2−/− and control mice. The bar chart represents the HSC frequency relative to untreated wild-type animals (n = 4 per group; ∗∗p < 0.01). (D) Representative flow cytometry plots of LKS and SLAM-LKS in whole bone marrow of Adh5+/−Fancd2−/− mice exposed to water or methanol. Data are represented as mean ± SEM. See also Figures S4 and S5.
Figure 5
Figure 5
DNA Damage Causes Liver Karyomegaly and Kidney Dysfunction in Adh5−/−Fancd2−/− Mice (A) H&E stain of liver sections from wild-type and Adh5−/−Fancd2−/− mice showing the central vein (400×). Scale bar represents 50 μm. (B) Quantification of hepatocyte nuclear DNA content (n = 3 mice per group; ∗p < 0.05; ∗∗p < 0.01). (C) Immunohistochemistry of liver sections from age-matched wild-type and Adh5−/−Fancd2−/− mice showing the presence of γ-H2A.X. Scale bar represents 50 μm. (D) Immunoblots for p53, γ-H2A.X, and histone H3 in nuclear extracts obtained from 4-week-old Adh5−/−Fancd2−/− mice and littermate controls. (E) Serum urea concentration in 5- to 6-week-old mice and congenic controls. Each point represents a single mouse (data are represented as mean ± SEM; ∗∗∗p < 0.001). (F) Urine (5 μl) obtained from individual mice was resolved by SDS-PAGE and stained with Coomassie blue. The urine obtained from Adh5−/−Fancd2−/− mice contains large amounts of a 60-kDa protein (∗, albumin). (G) Quantification of DNA content in kidney nuclei. Bar chart shows the percentage of nuclei that contain 2n or 4n DNA (n = 3 per group; ∗∗p < 0.01). (H) Panels show EM images of Adh5−/−Fancd2−/− animals and allelic controls, showing effacement of the foot processes in 6-week-old Adh5−/−Fancd2−/− mice (RBC, red blood cell; GBM, glomerular basement membrane; FP, podocyte foot processes; and U, urinary space). Scale bar represents 2 μm. See also Figure S6.
Figure 6
Figure 6
Consequences of Bone Marrow Transplantation in Adh5−/−Fancd2−/− Mice (A) Scheme outlining the protocol for the transplantation of wild-type bone marrow into Adh5−/−Fancd2−/− mice and their subsequent analysis. (B) Kaplan-Meier survival graph of non-transplanted and transplanted Adh5−/−Fancd2−/− mice. Blue/red squares denote mice developing T-cell leukemia and liver dysplasia, while the red square represents a mouse that developed both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). p < 0.0001 Log-Rank (Mantel-Cox test). (C) Serum aspartate transaminase (AST) levels as an indicator of liver function in the cohort of non-transplanted (inverted triangles) and transplanted Adh5−/−Fancd2−/− mice (squares). (D) Serum urea concentrations as an indicator of kidney function in the cohort of non-transplanted and transplanted Adh5−/−Fancd2−/− mice. Data are represented as mean ± SEM. (E) Left panels show EM images of 6-week-old and transplanted 28-week-old Adh5−/−Fancd2−/− mice (BMT, bone marrow transplant; GBM, glomerular basement membrane; FP, podocyte foot processes; and U, urinary space). Scale bar represents 2 μm. Right panel is a bar chart showing the quantification of foot process width (FPW, μm) from EM pictures. (F) Percentage of nuclei with two or more γ-H2A.X foci in kidney sections (∗p < 0.05). (G) Table showing the immunophenotype of the leukemic blasts in transplanted Adh5−/−Fancd2−/− mice. (H) Left panel, H&E staining of a liver section (400×) from the mouse showing both HCC and CC. N denotes normal hepatocytes. Right panels show H&E staining of liver sections (400×) from mice that developed leukemia but with abnormal hepatic histology (BDD, bile duct dysplasia; HD, hepatocyte dysplasia; L, leukemia; and N, normal liver). See also Figure S7.
Figure 7
Figure 7
Model for Genetic Protection against Endogenous Formaldehyde and Human Disease (A) In HSCs, two non-overlapping aldehyde catabolism systems operate to remove longer chained aldehydes (ALDH2) and formaldehyde (ADH5). Disruption of ADH5 has more drastic consequences on HSC function, possibly because the burden of formaldehyde is greater in HSCs compared to other aldehydes or because the former is more toxic. (B) Model integrating the DNA repair proteins that are known and that might protect against endogenous formaldehyde. Human genetic deficiency in DNA crosslink repair causes damage to three main organs. In FA, only the bone marrow is affected, in KIN only the kidney is affected, and in xeroderma pigmentosum/Cockayne syndrome (XPCS; variant Cockayne syndrome) all three organs are affected. Taking away ADH5 in FA-repair-defective mice results in damage in all organs such as what is seen in XPCS.
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