Embryonic Exposure to the Monoterpenes Thymol and p -Cymene Induces Social Deficits through KDM5 Inhibition

Abstract

Abstract The increasing prevalence of autism spectrum disorder (ASD) has drawn significant attention to its environmental origin. Environmental factors are estimated to contribute to 40% of ASD risk. However, the identities of these factors and their mechanisms of action remain poorly understood due to limitations of current research approaches. To bridge this gap, we screened a subset of the ToxCast library using a high-throughput social behavioral assay platform, Fishbook, to discover emerging ASD-relevant toxicants. From the 165 toxicants screened, we identified thymol (TH) and p-cymene (PC), both belonging to the chemical class monoterpenes, as potent inhibitors of social behavioral development through embryonic exposure. Monoterpenes are natural compounds commonly found in food flavorings, essential oils, and personal care products. Through a proteome-wide inverse virtual screening, followed by in silico, in vitro, and in vivo validations, we discovered lysine demethylase 5 (KDM5) family proteins, in particular KDM5B, KDM5C, and KDM5D, as predicted binding targets of TH and PC. Both KDM5B and KDM5C genes have been previously associated with ASD. The KDM5 family proteins are histone demethylases that selectively remove methyl groups from histone H3 lysine 4 (H3K4me3/2/1), which are epigenetic marks typically associated with active gene transcription. Both compounds directly inhibited KDM5 demethylase activity in nuclear extracts from zebrafish larvae and from human cells, and embryonic exposure increased the global level of H3K4me3 in vivo. Consistent with elevated H3K4me3, RNA-sequencing detected profound up-regulation of genes associated with genetic risk factors of ASD following TH and PC exposure. Furthermore, chemical inhibition of the opposing H3K4 methyltransferase complex, MLL/WDR5, effectively rescued social deficits induced by TH and PC. In summary, this work discovered thymol and p-cymene as risk factors for social behavioral development and establishes KDM5 inhibition as a mechanism of their neurodevelopmental toxicity.