Postdoctoral Scientist, MRC Laboratory of Molecular Biology
DNA is the blueprint of life, coding for all the information necessary to build an organism, maintain its integrity and pass the information from one generation to the next. However, thousands of DNA lesions arise in human cells every day. These lesions can corrupt the DNA information, leading to disease such as cancer. To prevent this, cells have evolved a machinery to repair DNA damage. However, these mechanisms are not completely efficient and some damage can still accumulate and cause disease. While significant advances have been made in the understanding of the mechanisms that repair damaged DNA, little is known about the factors that cause the damage in the first place.
Environmental sources of DNA damage, such as sun light or cigarette smoke, are widely recognized. It is far less appreciated that a substantial source of DNA damage is our own metabolism, which converts food and drink into building blocks but also generates molecules that damage DNA. One of these molecules is formaldehyde, a commonly used preservative and known carcinogen. Recently, we have shown that formaldehyde is generated within us in sufficient amounts to cause lethal DNA damage. However, we are protected from this threat by two systems: (1) formaldehyde detoxification and (2) DNA repair. When these two systems are inactivated in mice, DNA damage accumulates leading to cancer and dysfunction of vital organs.
These findings led me to investigate the origin of endogenous formaldehyde. Surprisingly, I found that a proportion of our endogenous formaldehyde comes from folate or vitamin B9. Folate is required to make some of the molecules that are crucial for life, such as DNA. This explains why folate is an essential nutrient, and why folate deficiency causes developmental abnormalities and anaemia. Folate needs to be consumed regularly, in part because this vitamin breaks apart in the body continuously due to spontaneous oxidation. In fact, folate supplements are given to pregnant women and some countries fortify certain foods with this vitamin. Surprisingly, I found that the spontaneous oxidation of folate can generate formaldehyde and damage DNA. Conversely, I then discovered that formaldehyde detoxification converts this carcinogen into an essential building block to make DNA. Thus, these findings reveal that folate and formaldehyde have two faces: a good one, because they support essential metabolism, and a bad one because they can damage DNA.
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.
Pub.: 29 Sep '15, Pinned: 31 Jul '17
Abstract: Reactive aldehydes are common carcinogens. They are also by-products of several metabolic pathways and, without enzymatic catabolism, may accumulate and cause DNA damage. Ethanol, which is metabolised to acetaldehyde, is both carcinogenic and teratogenic in humans. Here we find that the Fanconi anaemia DNA repair pathway counteracts acetaldehyde-induced genotoxicity in mice. Our results show that the acetaldehyde-catabolising enzyme Aldh2 is essential for the development of Fancd2(-/-) embryos. Nevertheless, acetaldehyde-catabolism-competent mothers (Aldh2(+/-)) can support the development of double-mutant (Aldh2(-/-)Fancd2(-/-)) mice. However, these embryos are unusually sensitive to ethanol exposure in utero, and ethanol consumption by postnatal double-deficient mice rapidly precipitates bone marrow failure. Lastly, Aldh2(-/-)Fancd2(-/-) mice spontaneously develop acute leukaemia. Acetaldehyde-mediated DNA damage may critically contribute to the genesis of fetal alcohol syndrome in fetuses, as well as to abnormal development, haematopoietic failure and cancer predisposition in Fanconi anaemia patients.
Pub.: 08 Jul '11, Pinned: 31 Jul '17
Abstract: Metabolism is predicted to generate formaldehyde, a toxic, simple, reactive aldehyde that can damage DNA. Here we report a synthetic lethal interaction in avian cells between ADH5, encoding the main formaldehyde-detoxifying enzyme, and the Fanconi anemia (FA) DNA-repair pathway. These results define a fundamental role for the combined action of formaldehyde catabolism and DNA cross-link repair in vertebrate cell survival.
Pub.: 15 Nov '11, Pinned: 31 Jul '17
Abstract: Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.
Pub.: 03 Jun '17, Pinned: 31 Jul '17
Abstract: Haematopoietic stem cells (HSCs) regenerate blood cells throughout the lifespan of an organism. With age, the functional quality of HSCs declines, partly owing to the accumulation of damaged DNA. However, the factors that damage DNA and the protective mechanisms that operate in these cells are poorly understood. We have recently shown that the Fanconi anaemia DNA-repair pathway counteracts the genotoxic effects of reactive aldehydes. Mice with combined inactivation of aldehyde catabolism (through Aldh2 knockout) and the Fanconi anaemia DNA-repair pathway (Fancd2 knockout) display developmental defects, a predisposition to leukaemia, and are susceptible to the toxic effects of ethanol-an exogenous source of acetaldehyde. Here we report that aged Aldh2(-/-) Fancd2(-/-) mutant mice that do not develop leukaemia spontaneously develop aplastic anaemia, with the concomitant accumulation of damaged DNA within the haematopoietic stem and progenitor cell (HSPC) pool. Unexpectedly, we find that only HSPCs, and not more mature blood precursors, require Aldh2 for protection against acetaldehyde toxicity. Additionally, the aldehyde-oxidizing activity of HSPCs, as measured by Aldefluor stain, is due to Aldh2 and correlates with this protection. Finally, there is more than a 600-fold reduction in the HSC pool of mice deficient in both Fanconi anaemia pathway-mediated DNA repair and acetaldehyde detoxification. Therefore, the emergence of bone marrow failure in Fanconi anaemia is probably due to aldehyde-mediated genotoxicity restricted to the HSPC pool. These findings identify a new link between endogenous reactive metabolites and DNA damage in HSCs, and define the protective mechanisms that counteract this threat.
Pub.: 28 Aug '12, Pinned: 31 Jul '17
Abstract: The early notion that cancer is caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. During the past decade, knowledge about the mechanisms by which genes erode and the molecular machinery designed to counteract this time-dependent genetic degeneration has increased markedly. At the same time, it has become apparent that inherited or acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer. This review summarizes the main DNA caretaking systems and their impact on genome stability and carcinogenesis.
Pub.: 18 May '01, Pinned: 31 Jul '17
Abstract: Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.
Pub.: 24 Jan '14, Pinned: 31 Jul '17
Abstract: One-carbon (1C) metabolism, mediated by the folate cofactor, supports multiple physiological processes. These include biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), epigenetic maintenance, and redox defense. Both within eukaryotic cells and across organs, 1C metabolic reactions are compartmentalized. Here we review the fundamentals of mammalian 1C metabolism, including the pathways active in different compartments, cell types, and biological states. Emphasis is given to recent discoveries enabled by modern genetics, analytical chemistry, and isotope tracing. An emerging theme is the biological importance of mitochondrial 1C reactions, both for producing 1C units that are exported to the cytosol and for making additional products, including glycine and NADPH. Increased clarity regarding differential folate pathway usage in cancer, stem cells, development, and adult physiology is reviewed and highlights new opportunities for selective therapeutic intervention.
Pub.: 20 Sep '16, Pinned: 31 Jul '17
Abstract: Folic acid fortification and supplementation has increased folate intake and blood folate concentrations and successfully reduced the incidence of neural tube defects. However, the developmental consequences of high folate intake are unknown. This study investigated the impact of high folate intake, alone or with methylenetetrahydrofolate reductase (MTHFR) deficiency, on embryonic and placental development in mice.Mthfr +/+ or +/- pregnant mice on a control diet (CD; recommended intake of folic acid for rodents) or folic acid-supplemented diet (FASD; 20-fold higher than the recommended intake) were examined for embryonic loss, delay, and defects at 10.5 and 14.5 days post coitum (dpc); 10.5-dpc placenta, and 14.5-dpc embryo hearts were studied histologically.Total plasma folate was 10-fold higher in FASD compared to CD mice; plasma homocysteine levels were not affected by diet. At 10.5 dpc, the FASD was associated with embryonic delay and growth retardation, and may confer susceptibility to embryonic defects. The FASD did not adversely affect 10.5-dpc placental development. At 14.5 dpc, embryos from the FASD Mthfr +/+ group were delayed and the FASD was associated with thinner ventricular walls in embryonic hearts. There was a significant interaction between maternal MTHFR deficiency and a high folate diet for several developmental outcomes.Our study suggests that high folate intake may have adverse effects on fetal mouse development and that maternal MTHFR deficiency may improve or rescue some of the adverse outcomes. These findings underscore the need for additional studies on the potential negative impact of high folate intake during pregnancy.
Pub.: 22 Jan '11, Pinned: 31 Jul '17
Abstract: Neural tube defects (NTDs), a common birth defect in humans, result from the failure of the embryonic neural tube (NT) to close properly. NT closure is a complex, poorly understood morphogenetic process influenced by genes and environment. The most effective environmental influence in decreasing the risk for NTDs is folic acid (FA) fortification and supplementation, and these findings led to the recommendation of periconceptual FA intake and mandatory fortification of the US grain supply in 1998. To explore the relationship between genetics and responsiveness to FA supplementation, we used five mouse NTDs models-Zic2, Shroom3, Frem2, Grhl2 (Grainyhead-like 2) and L3P (Line3P)-and a long-term generational FA supplementation scheme. Contrary to expectations, we find that three genetic mutants respond adversely to FA supplementation with increased incidence of NTDs in homozygous mutants, occurrence of NTDs in heterozygous embryos and embryonic lethality prior to NT closure. Because of these unexpected responses, we examined NTD risk after short-term FA supplementation. Our results indicate that, for the same genetic allele, NTD risk can depend on the length of FA exposure. Our data indicate that, depending on the gene mutation, FA supplementation may adversely influence embryonic development and NT closure.
Pub.: 23 Jun '11, Pinned: 31 Jul '17
Abstract: Folate catabolism involves cleavage of the C(9)-N(10) bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.
Pub.: 10 Nov '13, Pinned: 31 Jul '17
Abstract: Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors.
Pub.: 28 Oct '16, Pinned: 31 Jul '17
Abstract: Formaldehyde (FA) is a reactive signaling molecule that is continuously produced through a number of central biological pathways spanning epigenetics to one-carbon metabolism. On the other hand, aberrant, elevated levels of FA are implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for FA imaging are emerging as potentially powerful chemical tools to help disentangle the complexities of FA homeostasis and its physiological and pathological contributions. Currently available FA indicators require direct modification of the fluorophore backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other fluorophore classes. To address this challenge, we now present the rational, iterative development of a general reaction-based trigger utilizing 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, we developed a homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that is capable of masking a phenol on a fluorophore or any other potential chemical scaffold for related imaging and/or therapeutic applications. We demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular, Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red-shifted and FA sensitive in this series in terms of signal-to-noise responses and enables identification of alcohol dehydrogenase 5 (ADH5) as an enzyme that regulates FA metabolism in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and manipulating FA biology.
Pub.: 05 Apr '17, Pinned: 31 Jul '17
Abstract: Formaldehyde (FA) is a major reactive carbonyl species (RCS) that is naturally produced in living systems through a diverse array of cellular pathways that span from epigenetic regulation to the metabolic processing of endogenous metabolites. At the same time, however, aberrant elevations in FA levels contribute to pathologies ranging from cancer and diabetes to heart, liver, and neurodegenerative diseases. Disentangling the complex interplay between FA physiology and pathology motivates the development of chemical tools that can enable the selective detection of this RCS in biological environments with spatial and temporal fidelity. We report the design, synthesis, and biological evaluation of ratiometric formaldehyde probe (RFAP) indicators for the excitation-ratiometric fluorescence imaging of formaldehyde production in living systems. RFAP-1 and RFAP-2 utilize FA-dependent aza-Cope reactivity to convert an alkylamine-functionalized coumarin platform into its aldehyde congener with a ca. 50 nm shift in the excitation wavelength. The probes exhibit visible excitation and emission profiles, and high selectivity for FA over a variety of RCS and related reactive biological analytes, including acetaldehyde, with up to a 6-fold change in the fluorescence ratio. The RFAP indicators can be used to monitor changes in FA levels in biological samples by live-cell imaging and/or flow cytometry. Moreover, RFAP-2 is capable of visualizing differences in the resting FA levels between wild-type cells and models with a gene knockout of ADH5, a major FA-metabolizing enzyme, establishing the utility of this ratiometric detection platform for identifying and probing sources of FA fluxes in biology.
Pub.: 06 Jun '17, Pinned: 31 Jul '17
Abstract: Impairments in folate-mediated 1-carbon metabolism are associated with several common diseases and developmental anomalies including intestinal cancers, vascular disease, cognitive decline, and neural tube defects. The etiology of folate-associated pathologies involves interactions among multiple genetic risk alleles and environmental factors, although the causal mechanisms that define the role of folate and other B-vitamins in these complex disorders remain to be established. Folate and other B-vitamins fundamentally differ from other nutrients that interact with the genome in determining health and disease outcomes in that their interaction is reciprocal. Common gene variants influence the activity of folate-dependent enzymes and anabolic pathways; folate-mediated 1-carbon metabolism is essential for the high-fidelity synthesis of DNA and activated methyl groups that are required for DNA methylation and regulation of chromatin structure. This review focuses on the regulation of folate-mediated 1-carbon metabolism and its role in maintaining genome integrity and on strategies for establishing the metabolic pathways and mechanisms that underlie folate-associated pathologies.
Pub.: 09 Oct '09, Pinned: 31 Jul '17
Abstract: The recognition that mitochondria participate in folate-mediated one-carbon metabolism grew out of pioneering work beginning in the 1950s from the laboratories of D.M. Greenberg, C.G. Mackenzie, and G. Kikuchi. These studies revealed mitochondria as the site of oxidation of one-carbon donors such as serine, glycine, sarcosine, and dimethylglycine. Subsequent work from these laboratories and others demonstrated the participation of folate coenzymes and folate-dependent enzymes in these mitochondrial processes. Biochemical and molecular genetic approaches in the 1980s and 1990s identified many of the enzymes involved and revealed an interdependence of cytoplasmic and mitochondrial one-carbon metabolism. These studies led to the development of a model of eukaryotic one-carbon metabolism that comprises parallel cytosolic and mitochondrial pathways, connected by one-carbon donors such as serine, glycine, and formate. Sequencing of the human and other mammalian genomes has facilitated identification of the enzymes that participate in this intercompartmental one-carbon metabolism, and animal models are beginning to clarify the roles of the cytoplasmic and mitochondrial isozymes of these enzymes. Identifying the mitochondrial transporters for the one-carbon donors and elucidating how flux through these pathways is controlled are two areas ripe for exploration.
Pub.: 22 Jul '10, Pinned: 31 Jul '17