Sex-Related Effect of Chronic Doses of Warfarin and Menadione on Drosophila melanogaster

Vitamin K is a well-established cofactor for γ-glutamyl carboxylase in vertebrates, yet its physiological functions in insects remain poorly understood. Warfarin, a vitamin K antagonist, inhibits vitamin K epoxide reductase, thereby blocking the vitamin K cycle. We have previously demonstrated that, in Drosophila melanogaster, vitamin K3 (menadione sodium bisulfite, MSB) is converted into endogenous vitamin K2, and that warfarin interferes with this cycle, leading to reduced oxidative stress. In the present study, we investigated the effects of chronic exposure to MSB (3.5 mM) and/or warfarin (1 mM and 10 mM) during larval development on adult survival, lifespan, mitochondrial ATP hydrolysis rate, and the transcriptional expression of target genes. Warfarin at 1 mM did not affect development or egg laying, whereas 10 mM caused significant postembryonic lethality. Transcriptional analysis revealed marked sex-specific effects. In females, warfarin downregulated the levy gene (encoding a complex IV subunit) and the ATPsynB gene (encoding an ATP synthase subunit B), while MSB suppressed cell cycle genes (cmet, sti, mcm2). In contrast, males exhibited upregulation of COQ7 (encoding a key enzyme in ubiquinone biosynthesis) in response to warfarin and increased sti expression in response to MSB, suggesting a more robust compensatory response. Notably, the combined application of MSB and warfarin increased mitochondrial ATP hydrolysis, thereby offsetting the negative physiological effects despite persistent transcriptional suppression. In females, either MSB or warfarin alone significantly reduced median lifespan; however, when applied together, female lifespan was restored to control levels. In males, the effects were considerably weaker. Thus, this study reveals a novel, sex-specific interaction between vitamin K and its antagonist in D. melanogaster, providing new insights into mitochondrial adaptation and sexual dimorphism in metabolic stress responses.