AUTHOR=Lootens Orphélie , De Boevre Marthe , Gasthuys Elke , Van Bocxlaer Jan , Vermeulen An , De Saeger Sarah 

TITLE=Unravelling the pharmacokinetics of aflatoxin B1: In vitro determination of Michaelis–Menten constants, intrinsic clearance and the metabolic contribution of CYP1A2 and CYP3A4 in pooled human liver microsomes

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.988083

DOI=10.3389/fmicb.2022.988083

ISSN=1664-302X

ABSTRACT=<p>Mycotoxins, fungal secondary metabolites, are ubiquitously present in food commodities. Acute exposure to high levels or chronic exposure to low levels has an impact on the human body. The phase I metabolism in the human liver, performed by cytochrome P450 (CYP450) enzymes, is accountable for more than 80% of the overall metabolism of exogenous and endogenous compounds. Mycotoxins are (partially) metabolized by CYP450 enzymes. In this study, <italic>in vitro</italic> research was performed on CYP450 probes and aflatoxin B1 (AFB1), a carcinogenic mycotoxin, to obtain pharmacokinetic data on AFB1, required for further experimental work. The CYP450 probes of choice were a CYP3A4 substrate, midazolam (MDZ) and a CYP1A2 substrate, phenacetin (PH) since these are the main metabolizing phase I enzymes of AFB1. Linearity experiments were performed on the three substrates indicating that linear conditions were achieved at a microsomal protein concentration and incubation time of 0.25 mg/ml and 5 min, 0.50 mg/ml and 20 min and 0.25 mg/ml and 5 min for MDZ, PH and AFB1, respectively. The <italic>K<sub>m</sub></italic> was determined in human liver microsomes and was estimated at 2.15 μM for MDZ, 40.0 μM for PH and 40.9 μM for AFB1. The associated <italic>V</italic><sub>max</sub> values were 956 pmol/(mg.min) (MDZ), 856 pmol/(mg.min) (PH) and 11,536 pmol/(mg.min) (AFB1). Recombinant CYP systems were used to determine CYP450-specific Michaelis–Menten values for AFB1, leading to a CYP3A4 <italic>K<sub>m</sub></italic> of 49.6 μM and an intersystem extrapolation factor (ISEF) corrected <italic>V</italic><sub>max</sub> of 43.6 pmol/min/pmol P450 and a CYP1A2 <italic>K<sub>m</sub></italic> of 58.2 μM and an ISEF corrected <italic>V</italic><sub>max</sub> of 283 pmol/min/pmol P450. An activity adjustment factor (AAF) was calculated to account for differences between microsome batches and was used as a correction factor in the determination of the human <italic>in vivo</italic> hepatic clearance for MDZ, PH and AFB1. The hepatic blood clearance corrected for the AAF CL<sub>H,B,MDZ,AAF</sub>, CL<sub>H,B,PH,AAF</sub> CL<sub>H,B,AFB1,AAF(CYP3A4)</sub> and CL<sub>H,B,AFB1,AAF(CYP1A2)</sub> were determined in HLM at 44.1 L/h, 21.7 L/h, 40.0 L/h and 38.5 L/h. Finally, inhibition assays in HLM showed that 45% of the AFB1 metabolism was performed by CYP3A4/3A5 enzymes and 49% by CYP1A2 enzymes.</p>