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

Abstract

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 Km 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 Vmax 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 Km of 49.6 μM and an intersystem extrapolation factor (ISEF) corrected Vmax of 43.6 pmol/min/pmol P450 and a CYP1A2 Km of 58.2 μM and an ISEF corrected Vmax 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.

Exploring the Impact of Efavirenz on Aflatoxin B1 Metabolism: Insights from a Physiologically Based Pharmacokinetic Model and a Human Liver Microsome Study

“…The formation of AFB1 and AFM1 radicals that react with proteins in the sample is also hypothesized [ 30 ]. Currently, research has been performed on the metabolism of AFB1, both in vitro and in vivo, but more research is a prerequisite to fully assign the observed AFB1 decrease to metabolites [ 25 , 26 , 31 ]. Understanding these interactions is crucial, especially for HIV patients consuming AFB1-contaminated food, as EFV could influence AFB1 immunotoxicity.…”

“…Samples were prepared based on the protocol detailed in Lootens et al, 2022 [ 31 ]. In brief, centrifugal Eppendorf cups were filled with 50 µL substrate solution (i.e., 5 µM for AFB1 or 5 µM for MDZ) and with 50 µL of 1.15% KCl or 5 µL of co-substrate EFV (7.9 µM or 250 µM) and 45 µL of 1.15% KCl solution, in case of co-incubation.…”

Building a Human Physiologically Based Pharmacokinetic Model for Aflatoxin B1 to Simulate Interactions with Drugs

“…In vitro–in vivo extrapolation (IVIVE) is, therefore, the ultimate tool: by performing in vitro experiments on human cells or microsomes, one can determine the metabolic parameters needed to build a PBPK model. In this case, not much data were available for AFB1; therefore, in vitro experiments were performed and certain parameters were predicted based on the physicochemical characteristics of AFB1 [ 31 ]. The model was built using a bottom-up approach without any fitting.…”

Possible Mechanisms of the Interplay between Drugs and Mycotoxins—Is There a Possible Impact?

“…Noteworthy, there was a triphasic effect observed in the 500 mg OPZ group, where no effect was observed in the first month, a decrease in AFB1-adduct formation occurred in the second month of OPZ intake and in the first month post-intervention [87]. Recently, PK parameters of AFB1 were unraveled by Lootens et al, (2022) giving more insight in what happens with AFB1 in the human body [97]. It is clear that more research needs to be performed both in vitro and in vivo to have a better insight in possible interactions between mycotoxins and other compounds, such as drugs.…”