4.6 Article

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, Issue -, Pages -

Publisher

FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2022.988083

Keywords

mycotoxins; CYP450 enzymes; pharmacokinetic; in vitro; Michaelis-Menten constant; LC-MS; MS; aflatoxin B1; human liver microsomes

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Mycotoxins, which are fungal secondary metabolites, have a widespread presence in food and can have harmful effects on the human body. This study focused on the in vitro interaction between CYP450 probes and the carcinogenic mycotoxin aflatoxin B1 (AFB1) and obtained pharmacokinetic data on AFB1. The findings contribute to further research on the metabolism of mycotoxins and their impact on human health.
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, in vitro 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 K-m was determined in human liver microsomes and was estimated at 2.15 mu M for MDZ, 40.0 mu M for PH and 40.9 mu M for AFB1. The associated V-max 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 K-m of 49.6 mu M and an intersystem extrapolation factor (ISEF) corrected V-max of 43.6 pmol/min/pmol P450 and a CYP1A2 K-m of 58.2 mu M and an ISEF corrected V-max 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 in vivo hepatic clearance for MDZ, PH and AFB1. The hepatic blood clearance corrected for the AAF CLH,B,MDZ,AAF, CLH,B,PH,AAF CLH,B,AFB1,AAF(CYP3A4) and CLH,B,AFB1,AAF(CYP1A2) 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.

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