Key Takeaways & Executive Findings
- •• • 9-Fluorenone exposure at 10 and 30 μmol·L−1 significantly increased HepG2 cell proliferation (colony formation and EdU staining), indicating a dose-dependent tumor-promoting effect. • • Nr1d1 mRNA expression was suppressed by 9-fluorenone in a dose-dependent manner, with a notable reduction in oscillation amplitude in synchronized cells, implicating circadian disruption. • • Pretreatment with the Nr1d1 agonist SR9009 effectively blocked 9-fluorenone-induced proliferation, confirming Nr1d1 as a key mediator and potential therapeutic target. • • The study establishes a mechanistic link between environmental OPAH exposure and clock gene dysregulation, highlighting Nr1d1 as a biomarker for OPAH-induced carcinogenesis.
Abstract
Oxygenated polycyclic aromatic hydrocarbons (OPAHs) are prevalent environmental contaminants with high toxicity and demonstrated tumor-promoting effects. Circadian clock genes are critical regulators of cellular homeostasis and tumorigenesis. This study investigated the role of clock genes in OPAH-induced tumor promotion using 9-fluorenone (FLO), a dominant OPAH. Human hepatocellular carcinoma (HepG2) cells were exposed to 0, 10, and 30 μmol·L−1 FLO. Cell proliferation was assessed via colony formation and EdU staining, and mRNA expression of core clock genes (Bmal1, Npas2, Nr1d1, Per2, Cry1, Dbp) was quantified by RT-qPCR. Results demonstrated that FLO exposure significantly enhanced cell proliferation and dose-dependently suppressed Nr1d1 expression. In synchronized cells, FLO dampened the amplitude of Nr1d1 mRNA oscillation. Pretreatment with SR9009, a selective Nr1d1 agonist, effectively inhibited FLO-induced proliferation. These findings indicate that Nr1d1 plays a pivotal role in the tumorigenic cascade initiated by polycyclic aromatic hydrocarbon derivatives, providing mechanistic insights into OPAH health impacts and potential intervention strategies.
1. Introduction
Oxygenated polycyclic aromatic hydrocarbons (OPAHs) are ubiquitous environmental pollutants arising from incomplete combustion and photochemical transformation of parent PAHs. Their concentrations in air, water, and soil often rival or exceed those of their precursors, yet their toxicological profiles remain poorly characterized. Among OPAHs, 9-fluorenone (FLO) is particularly abundant and has been detected at high levels in atmospheric particulate matter and groundwater. While the tumor-promoting effects of OPAHs are recognized, the molecular mechanisms—especially the involvement of circadian clock genes—are largely unexplored. This knowledge gap hampers accurate risk assessment and the development of targeted preventive strategies.
This study addresses the bottleneck by systematically evaluating the impact of FLO on the expression of core clock genes in HepG2 cells, a model of hepatocellular carcinoma. By employing colony formation and EdU assays alongside RT-qPCR, we demonstrate that FLO enhances proliferation while specifically suppressing Nr1d1, a key circadian repressor. The use of the Nr1d1 agonist SR9009 further validates the functional relevance of this pathway. These findings not only elucidate a novel mechanism of OPAH toxicity but also propose Nr1d1 as a potential therapeutic target for environmental carcinogenesis.
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MA Jiangsong, JIANG Zihao, GAO Rui (2026). Regulatory Role of the Clock Gene Nr1d1 in Oxygenated Polycyclic Aromatic Hydrocarbons 9-Fluorenone Induced Tumorigenesis. Environmental Chemistry. https://doi.org/10.7524/j.issn.0254-6108.2025010307
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Frequently Asked Questions
What is the significance of the dose-dependent suppression of Nr1d1 by 9-fluorenone in the context of circadian disruption and cancer progression?
The dose-dependent suppression of Nr1d1 mRNA expression (at 10 and 30 μmol·L−1) indicates that FLO disrupts the circadian transcriptional network. Nr1d1 is a core negative limb of the circadian clock, and its downregulation can lead to uncontrolled cell proliferation. This provides a mechanistic link between environmental pollutant exposure and circadian-related tumorigenesis.
How does the use of SR9009, a Nr1d1 agonist, validate the role of Nr1d1 in FLO-induced proliferation?
Pretreatment with SR9009 effectively inhibited FLO-induced cell proliferation, as shown by reduced colony formation and EdU incorporation. This rescue experiment confirms that Nr1d1 activation counteracts the proliferative effects of FLO, establishing a causal relationship and suggesting that Nr1d1 agonists could be potential therapeutic agents.
What are the implications of the reduced Nr1d1 oscillation amplitude in synchronized cells for understanding chronodisruption?
The dampened amplitude of Nr1d1 mRNA oscillation in synchronized cells indicates that FLO disrupts the circadian rhythm at the molecular level. This chronodisruption may contribute to the loss of temporal control over cell division, thereby promoting tumorigenesis. It underscores the importance of circadian integrity in cancer prevention.
Could the findings be extrapolated to other OPAHs or cell types, and what are the limitations?
While this study focused on FLO and HepG2 cells, the mechanistic pathway involving Nr1d1 may be relevant to other OPAHs and cancer types. However, extrapolation requires caution due to potential differences in metabolism and tissue-specific clock regulation. Further studies with additional OPAHs and in vivo models are necessary to establish generalizability.
What are the potential clinical or environmental health applications of this research?
The identification of Nr1d1 as a key mediator of OPAH-induced proliferation offers a potential biomarker for early detection of exposure effects and a target for chemoprevention. Additionally, the study supports the use of circadian-modulating agents like SR9009 in mitigating the carcinogenic risks of environmental pollutants, informing public health policies and therapeutic strategies.
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