*3.2. Role of Circulatory CYPs in Drug Metabolism and in Cell–Cell Communication*

Produced by and secreted from cells into extracellular plasma, EVs transmit genetic material, proteins, and other biological cargos that reflects the function of the organ from which they originate [24,81]. Once EVs exit the cell by exocytosis, they travel to distant cells via biological fluids such as plasma, cerebrospinal fluid, and urine, where they fuse with recipient cells [81]. The cargo is then released and is free to exert its effects on target cells [81,82]. This transmission of EVs throughout the body provides a means of communication between cells—offering a new source of biomarkers, as well as a potential tool in characterizing variability in drug exposure and therapeutic intervention [24,82].

Furthermore, studies have revealed that EVs carry a multitude of drug metabolizing enzymes, including members of the CYP enzyme group [23,83,84] (Table 2). However, the presence and amount of CYP enzymes is likely to vary greatly depending on the EV source, whether the EVs are isolated from plasma or a specific cell line, as well as the physiological condition of the cells from which they originate [85].


**Table 2.** Select xenobiotic metabolizing cytochrome P450 enzymes expressed in EVs.

Key: + mRNA, ++ protein, +++ activit.

Our group recently detected CYPs 1B1, 2A6, 2E1, and 3A4 mRNA in plasma-derived EVs from healthy subjects, with 2E1 displaying > 500-fold higher expression than the other CYPs identified. We also detected CYPs 1A1, 1B1, 2A6, 2E1, and 3A4 at the protein level [23]. In our studies, plasma EVs were isolated using 0.22 μm filtration, followed by different methods including single and double isolations with a commercial kit [23,26], in addition to the ultracentrifugation method [92]. Further, absolute spectra revealed a higher level of CYPs in plasma-derived EVs versus plasma alone, which indicates specific packaging of CYPs within circulating plasma EVs. Importantly, activity assays confirmed the enzymatic activity of EV CYP2E1 and 3A4. Interestingly, our finding indicated a higher level of CYP2E1 in plasma EVs than in liver cells/EVs, which is the powerhouse of CYP enzymes. The plasma EV CYP2E1 level was also higher than alcohol-induced CYP2E1 in monocytes. Together, these findings suggest that EV packaging is carefully regulated. A study performed by Rowland et al. further strengthened our findings, demonstrating the presence of peptides and mRNA of CYPs, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2 J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH-cytochrome CYP reductase in plasma-derived exosomes [86]. As EVs act as intercellular messengers, this differential packaging has a crucial impact on the pathophysiology of the recipient cells, and an abundance of CYP enzymes in EVs suggests their necessity at points across the body.
