Abstract Background: Formaldehyde, a human carcinogen found in industrial chemicals, is also produced by mammalian metabolism. The toxicity of endogenous formaldehyde is most severe in children with inborn errors in formaldehyde detoxification enzymes (ALDH2/ADH5), causing early-onset bone marrow failure and leukemia through formaldehyde-DNA damage. While endogenous formaldehyde exists in all healthy tissues, the nutritional and metabolic factors controlling its production remain unknown. Prior in vitro studies showed folate can spontaneously decompose to release formaldehyde. However, folate metabolism's contribution to endogenous formaldehyde in vivo has never been studied. Given widespread folate supplementation and the 2 billion people with folate insufficiency worldwide, we investigated how folate excess and deficiency affect endogenous formaldehyde in mouse models. Methods: For folate excess, wildtype and Adh5-/- mice received high-folate diet (10x recommended daily allowance) for 8 weeks. For deficiency: 1) SLC46A1 deletion causing folate malabsorption, and 2) folate-depleted diet for 8-12 weeks. We developed ultra-sensitive mass spectrometry to quantify formaldehyde-DNA adducts as biomarkers of tissue formaldehyde exposure. Results: Contrary to in vitro predictions, tissue folate accumulation did not elevate endogenous formaldehyde. Strikingly, folate deficiency significantly increased formaldehyde in liver, spleen, and bone marrow. In folate-deficient Adh5-/- mice with impaired formaldehyde clearance, hepatic formaldehyde increased 35-fold with elevated DNA damage, severe anemia (50% reduction in red cells), and hematopoietic stem cell loss. Metabolomics revealed folate-deficient livers upregulated choline oxidation to generate one-carbon units, a pathway that produces formaldehyde as a toxic byproduct. Conclusions: We establish folate metabolism as a critical safeguard against endogenous formaldehyde accumulation, with deficiency triggering formaldehyde production through compensatory choline oxidation. Our findings have three major implications: First, they validate the safety of folate supplementation, used by millions for prenatal care and cancer treatment, by proving excess folate does not generate formaldehyde. Second, we hypothesize a mechanism underlying cancer risk in folate-deficient individuals worldwide, particularly the 500 million with ALDH2 deficiency who cannot detoxify formaldehyde. Third, we identify choline as an unexpected endogenous formaldehyde source, highlighting the need for caution with choline supplementation during low-folate status. These findings support targeted folate supplementation as a cancer prevention strategy for high-risk populations with ALDH2 deficiency Citation Format: Christopher Mellor, Saeideh Azad, Nicholas Cheng, Brandon James, Vicky A. Simon, Olga V. Malysheva, Guillermo Burgos Barragan, John Blenis, Martha S. Field, Meng Wang. Nutritional disruption of folate homeostasis triggers an endogenous genotoxic carcinogen abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 935.
Mellor et al. (Fri,) studied this question.