Unraveling Paracetamol Metabolism and Its Circadian Regulation: Insights from Tobacco Hairy Roots as a Model System

The increasing interest in the presence of contaminants of emerging concern (CEC) in aquatic environments has driven research into biological mechanisms capable of eliminating pharmaceutical compounds like paracetamol, considering different plant species as model systems. Thus, the use of hairy roots (HRs) has become an interesting tool. This study explores the ability of tobacco HRs to remove paracetamol, with an emphasis on elucidating the main metabolism steps and key enzymes involved in the green liver detoxification process, as well as the antioxidant response. The deepening of these aspects has been carried out through gene expression and biochemical analysis under circadian regulation. Our results reveal that HRs efficiently removed paracetamol (100 mg L^-1) from the culture medium, achieving around 99% removal at ZT16 h (<i>Zeitgeber Time</i> 16). The early activation of antioxidant defense mechanisms, demonstrated by enhanced peroxidase (POD) activity and total antioxidant capacity (TAA) during the light phase, has been observed. Furthermore, glutathione S-transferase (GSTs) activity and glutathione (GSH) levels, potentially linked to paracetamol conjugation, were also assessed. Gene expression analyses confirmed GST gene upregulation in response to paracetamol treatment, with <i>GSTF6-like</i> and <i>GSTF8-like</i> maintaining circadian rhythms as in the control, and <i>GSTZ1-like</i> only displayed rhythmic expression upon treatment. Additionally, the modulation of core circadian clock genes (<i>NtLHY1</i> and <i>NtTOC1</i>) suggests that the plant response to paracetamol is tightly regulated by the circadian system. Together, these findings shed light on the complex molecular and biochemical mechanisms underlying paracetamol detoxification in tobacco HRs and underscore the significant role of circadian regulation in orchestrating these responses.