Identification of toxic substances considering circadian disruption: an integrated approach combining reduced transcriptomic analysis, behavioral monitoring, and time-course gene expression profiling

The circadian clock coordinates daily oscillations of essential physiological and behavioral processes in organisms. Conversely, aberrant circadian rhythms, triggered by external cues such as circadian disruptive compounds, have been linked with various chronic diseases and aging. To search for environmental molecules that perturb circadian rhythms, we conducted a high-throughput screening of environmental compounds utilizing an integrated, multi-tiered approach combining reduced transcriptomic analysis, behavioral monitoring, and time-course gene expression profiling. This strategy enabled us to analyze 84 environmental compounds, covering over 750 gene expression signatures, and prioritized compounds with significant circadian disruptive potentials. We identified 16 compounds that exerted the most pronounced transcriptional effects on circadian system, with steroid hormones (e.g. dexamethasone and triamcinolone), biocides (triclosan and prochloraz), and metallic compounds (AgNO₃ and AgNPs), emerging as the key classes of circadian disruptors. Functional enrichment analyses revealed that these compounds modulate core circadian regulatory networks (e.g. per, cry, nr1d, bmal families) and downstream biological processes such as transcriptional regulation, mRNA splicing, and metabolic homeostasis. In addition, behavioral assays conducted for eleven representative compounds and time-course gene expression profiling performed for three compounds further demonstrated significant alterations in locomotor activity rhythms and molecular circadian dynamics, including dihydrotestosterone, prochloraz and AgNO₃, emphasizing their disruptive effects at both physiological and transcriptional levels. Collectively, the circadian disruptors identified in this study provide novel insights into the potential risks of these compounds to ecological and human health.