Vitamin E Deficiency Causes Mortality in Zebrafish Embryos via Metabolic Dysregulation Due to Redox‐Mediated Mechanisms
Authors: McDougall Melissa Q , Choi Jaewoo , Kim Hye‐Kyeong , Bobe Gerd , Ho Emily , Stevens Jan Frederik , Cadenas Enrique , Tanguay Robert , Traber Maret G. 
Published: November 2023

Vitamin E (α‐tocopherol; VitE), a lipophilic antioxidant, prevents fetal resorption in VitE− deficient pregnant rodents through unknown mechanisms. We sought to determine the cause of mortality during developmental VitE deficiency using an embryonic zebrafish model. Adult 5D zebrafish fed defined diets without (E−) or with VitE (E+, 500 mg/kg diet RRR‐α‐tocopheryl acetate) were spawned to obtain E− and E+ embryos that were evaluated up to 120 hours‐post‐fertilization (hpf). At 48 hpf, E− embryos began showing deformities and >75% were dead or malformed by 120 hpf. This morbidity and mortality time‐course in E− embryos paralleled depletion of total and free (unesterified) docosahexaenoic acid (DHA; each p<0.001) with concomitant increases in autoxidized DHA derivatives, including 7‐ and 10‐hydroxy DHAs (each p<0.001), compared to E+ embryos. Lipid peroxidation in E− embryos coincided with perturbations in antioxidant status (e.g. decreased ascorbic acid and glutathione) and depletion of NADPH (each p<0.001). VitE deficiency also induced a secondary choline deficiency (p= 0.0316) that consequently decreased levels of methyl‐donors (e.g. S‐adenosylmethionine; p<0.001) and disrupted DNA methylation, as shown by reduced dmnt1 expression (p<0.001) and global DNA hypo‐methylation (p= 0.003 for VitE condition), from 24 to 120 hpf in E− vs. E+ embryos. Additionally, VitE deficiency caused mitochondrial bioenergetic perturbations resembling the Warburg effect: at 24 hpf E− embryos had elevated levels of glucose (p<0.001) and tricarboxylic acid cycle intermediates (all p<0.01) that were depleted by 72 hpf, relative to E+ embryos, yet maintained higher levels of lactate (p<0.001) throughout development. Corresponding results in E− vs. E+ embryos using assays of oxygen consumption rate [OCR] and proton production rate [PPR] revealed E− embryos were hyper‐metabolic at 24 hpf but had decreased basal (p<0.001) and maximal (p<0.001) OCRs and a two‐fold increased PPR (p<0.001) by 48 hpf. Glucose injection into the yolk at 24 hpf partially attenuated morbidity and mortality in 120 hpf E− embryos. This body of data suggests VitE deficiency‐induced hyper‐metabolism and subsequent glucose depletion causes lethal metabolic dysfunction in E− embryos, presumably due to perturbations in both (a) the cellular antioxidant network, which shift NADPH towards repair of oxidative damage instead of aerobic energy metabolism, and (b) methyl‐donor status, with ensuing global DNA hypo‐methylation. In summary, we show novel evidence that VitE represents a pleiotropic nexus for the redox‐modulation of metabolism, thus providing a mechanistic rationale for VitE deficiency‐induced embryonic mortality.Support or Funding InformationNational Institutes of Health Grants S10RR027878 (MGT and JFS) and NIEHS P30 ES000201 (RT) supported this work. MM was supported in part by National Science Foundation Grant DGE 0965820. H‐KK sabbatical support provided by The Catholic University of Korea. MGT supported in part by the Helen P Rumbel endowment to the Linus Pauling Institute of Oregon State University.