In vitro enantioselective study of the toxicokinetic effects of chiral fungicide tebuconazole in human liver microsomes

Ecotoxicology and Environmental Safety 181 (2019) 96–105

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In vitro enantioselective study of the toxicokinetic effects of chiral fungicide tebuconazole in human liver microsomes

T

Maísa Daniela Habenschusa, Viviani Nardinia, Luís Gustavo Diasa, Bruno Alves Rochab, Fernando Barbosa Jr.b, Anderson Rodrigo Moraes de Oliveiraa,∗ a

Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil Laboratório de Toxicologia e Essencialidade de Metais, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14049-903, Ribeirão Preto, SP, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tebuconazole 1-Hydroxytebuconazole Pesticide Enantioselective In vitro metabolism Toxicokinetic prediction

Tebuconazole (TEB) is a chiral triazole fungicide that is globally marketed and used as a racemic mixture to control plant pathogens. Due to its use as a racemic mixture, TEB may exhibit enantioselective toxicokinetics toward nontarget organisms, including humans. Therefore, the in vitro enantioselective metabolism of TEB by cytochrome P450 enzymes (CYP450) was studied using human liver microsomes, and the in vivo toxicokinetic parameters were predicted. A new enantioselective, reversed-phase LC-MS/MS method was developed and validated to analyze the enantiomers of TEB and its main metabolite, 1-hydroxytebuconazole (TEBOH). In vitro metabolic parameters were obtained, and in vitro-in vivo extrapolations were performed. Michaelis-Menten and atypical biphasic kinetic profiles were observed with a total intrinsic clearance ranging from 53 to 19 mL min−1 mg−1. The in vitro-in vivo extrapolation results showed that TEB first passage effect by the liver seems to be negligible, with hepatic clearance and extraction ratios ranging from 0.53 to 5.0 mL min−1 kg−1 and 2.7–25%, respectively. Preferential metabolism of (+)-TEB to rac-TEB and (−)-TEB was observed, with preferential production of (+)-TEBOH. Furthermore, reaction phenotyping studies revealed that, despite the low hepatic clearance in the first pass metabolism of TEB, multiple human CYP450 isoforms were involved in TEB metabolism when TEBOH enantiomers were generated, mainly CYP3A4 and CYP2C9, which makes TEB accumulation in the human body more difficult due to multiple metabolic pathways.

1. Introduction Tebuconazole (TEB, Fig. S1A) ((RS)-1-(4-chlorophenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-pentan3-ol) is a globally marketed triazole systemic fungicide that is used to control plant pathogens and acts by inhibiting the fungal enzyme, lanosterol-14α-demethylase (CYP51). Despite TEB being considered safe for agricultural production under regulatory conditions, TEB is classified as a potential human carcinogen (C rating, USEPA, 2016), is suspected of damaging fetuses (European Food Safety Authority, 2014) and affecting human pregnancy (Zhou et al., 2016) and has potential action as an androgenic disruptor (Lv et al., 2017). People are constantly exposed to TEB through either diet (Clasen et al., 2018; Cotton et al., 2016; Freeman et al., 2016; Herrero-Hernández et al., 2013; Saitta et al., 2017) or occupational exposure, especially agricultural workers (Fustinoni et al., 2014; Zhou et al., 2016), and the known TEB metabolic pathway in

humans involves, first t-butyl group hydroxylation to produce 1-hydroxytebuconazole (TEBOH, Fig. S1B) ((RS)-5-(4 chlorophenyl-2,2-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-1,3 pentanediol) (Mercadante et al., 2014). TEB and TEBOH are chiral compounds, and their enantiomers have distinct behavior in nontarget organisms. TEB, for example, has already shown enantioselectivity in different investigations, such as fungicidal activity and accumulation (Liu et al., 2016), toxicity (Li et al., 2015), soil dissipation (Wang et al., 2012), tissue and plasma degradation (Zhu et al., 2007) and animal liver metabolism (Shen et al., 2012). Enantioselective TEB behavior in non-target organisms and risks posed by human exposure to this fungicide highlight the importance of examining the enantioselective toxicokinetics of TEB in humans (de Albuquerque et al., 2018). Toxicokinetic data are most often obtained from in vivo animal studies measuring plasma or tissue concentrations of a xenobiotic over time. However, humans differ from animals in

∗ Corresponding author. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP, Av. dos Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14040-901, Brazil. E-mail address: [email protected] (A.R.M. de Oliveira).

https://doi.org/10.1016/j.ecoenv.2019.05.071 Received 9 January 2019; Received in revised form 21 May 2019; Accepted 25 May 2019 Available online 05 June 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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obtained during the HPLC-DAD screening procedure were used to develop the enantioselective method by liquid chromatography tandem mass spectrometry (LC-MS/MS). TEB and TEBOH enantiomers were detected and quantified on a Thermo Scientific LC system equipped with a pump (Accela 600 pump) and an autosampler (Accela autosampler) coupled with a Thermo Scientific TSQ Quantum Access Max electrospray triple quadrupole mass spectrometer operating in positive mode. Chromatographic separation was carried out on a Chiralcel OJ® column (250 × 4.6 mm, 10 μm) using methanol:water (90:10, v/v) as the mobile phase at a flow rate of 0.6 mL min−1 and a column temperature of 25 °C. The injection volume was 10 μL, and the sample tray temperature was kept at 10 °C. rac-TEB, rac-TEBOH, and IS (all prepared at 1 mg L−1 in methanol:water, 90:10, v/v) were directly infused at a flow rate of 0.03 mL min−1 and, after equipment self-adjustment, capillary voltage was set at + 5000 V and capillary temperature was set at 320 °C. Nitrogen was used as the sheath gas and auxiliary gas at flow rates of 10 and 5 (arbitrary units), respectively, and the vaporizer temperature was set at 290 °C. Argon was used as a collision-induced dissociation gas at 2.1 mTorr. Multiple reaction monitoring transitions were selected, and two reactions were used for quantification (Q) and confirmation (C), with their respective collision energies (CE) at m/z 308 → 70Q and 308 → 125C (CE 21 and 33 V) for TEB enantiomers, m/z 324 → 70Q and 324 → 125C (CE 21 and 40 V) for TEBOH enantiomers, and m/z 304 → 217Q and 304 → 202C (CE 23 and 34 V) for IS. Xcalibur software version 2.0 (Thermo Fisher Scientific) was used to control instruments and to process data. Finally, the enantioselective LC-MS/MS method developed was validated based on European Medicines Agency (EMA) guidelines for Bioanalytical Method Validation (European Medicines Agency, 2012). The evaluated parameters were linearity, selectivity, carryover, lower limit of quantification, precision and accuracy, matrix effect and stability. In addition, the racemization of isolated TEB enantiomers under incubation conditions was evaluated (see Supplementary Material).

numerous ways, and the use of in vivo animal data are being criticized due to poor correlation with the actual effects observed in humans (Punt et al., 2017). In vitro toxicokinetic data obtained using humanderived models, on the other hand, have been demonstrated to adequately predict thorough in vitro-in vivo extrapolation methods. They are being increasingly recognized as potential tools for evaluating chemical toxicokinetics in humans (Abass, 2013). Therefore, to improve risk assessment of human exposure to the chiral fungicide, tebuconazole, this paper evaluated the in vitro enantioselective TEB metabolism by Human Liver Microsomes (HLM) by monitoring enantioselective production of the main hydroxylated metabolite, TEBOH, a chiral compound that has already been found in human urine samples (Mercadante et al., 2014). To this, an enantioselective reversed-phase LC-MS/MS method was developed and validated for TEB and TEBOH enantiomer analysis in a matrix composed of HLM. rac-TEB, (+)-TEB, and (−)-TEB were individually incubated and the TEBOH formation rate was determined. Kinetic profiles were evaluated individually, in vitro-in vivo toxicokinetic extrapolations were performed and the major CYP450 isoforms involved in TEB metabolism were identified. 2. Materials and methods 2.1. Chemicals and reagents Rac-tebuconazole (rac-TEB, ≥ 99%) and fenamiphos (FEN, ≥ 96%), used as an internal standard (IS), were acquired from SigmaAldrich (St. Louis, MO, USA). The rac-1-hydroxytebuconazole (racTEBOH, ≥ 98.5%) was acquired from Toronto Research Chemicals (Toronto, Canada). Standard TEB and TEBOH stock solutions were prepared in methanol (6800 μmol L−1 and 72 μmol L−1, respectively). Standard FEN stock solution was prepared in acetonitrile (21 μmol L−1). Pooled human liver microsomes and recombinant human CYP450 isoforms (rCYPs) were supplied by Corning Life Science (Phoenix, AZ, USA) and stored at −80 °C. Other reagents, such as chemical inhibitors used in CYP450 reaction phenotyping studies, are presented in the Supplementary Material.

2.4. Optical rotation sign and elution order Circular dichroism (CD) (Jasco J-810, Tokyo, Japan) analyses were carried out to determine the optical rotation sign for the isolated TEB and TEBOH enantiomers. Enantiomers were solubilized in the mobile phase, methanol: water (90:10, v/v), and spectra were acquired from 190 to 260 nm at 25 °C after correction with a blank solution. A cell with a path length of 1 mm was employed, and four scans were accumulated. After optical rotation sign, each enantiomer was analyzed by the developed LC-MS/MS method (section 2.3) to confirm the elution order.

2.2. Tebuconazole and 1-hydroxytebuconazole enantiomers isolation TEB and TEBOH enantiomers were isolated using a Shimadzu highperformance liquid chromatography coupled with diode array detection (HPLC-DAD) system (Kyoto, Japan) consisting of two LC-10AD solvent pump units, a DGU-20A3 degasser and SCL-10AVP system controller. The diode array detector, SPD-M20A, was operated at 220 nm. Isolation was carried out at room temperature (20 ± 2 °C). A Lux Amylose-2® (150 × 4.6 mm, 5 μm) column acquired from Phenomenex (Torrance, CA, USA) was employed, and the mobile phase comprised hexane:isopropanol (85: 15, v/v) at a flow rate of 1 mL min−1. Manual sequential injections of 50 μL of standard rac-TEB (5 mg mL−1) and racTEBOH (1 mg mL−1) stock solutions were conducted, separately, and data were analyzed with LC solution software, 1.25 SP1 (Shimadzu, Kyoto, Japan). TEB and TEBOH enantiomers were individually collected, and the mobile phase was evaporated under a gentle compressed air stream. Finally, the isolated enantiomers were solubilized in methanol and stored in amber glass tubes at −20 °C for further identification (section 2.4).

2.5. In vitro metabolism incubation conditions In general, incubation medium (n = 3) for the in vitro metabolism studies consisted of the substrate (rac-TEB, (−)-TEB, or (+)-TEB), phosphate buffer (100 mmol L−1, pH 7.4), NADPH regenerating system (NADP+ at 0.25 mmol L−1, D-glucose-6-phosphate at 5.0 mmol L−1, and glucose-6-phosphate dehydrogenase at 0.4 U mL−1) and HLM (0.2 mg mL−1) or rCYPs (50 pmol L−1) in a final volume of 200 μL. Samples were prewarmed in a water shaker bath (Dubnoff Metabolic Shaking Incubator SL157, Solab, Brazil) at 37 °C for 5 min, and metabolism reactions were initiated by addition of NADPH regenerating system. Controls (n = 3), without the addition of NADPH regenerating system, were also prepared. After 20 min of incubation, reactions were stopped by addition of 1 mL of ethyl acetate and 30 μL of IS. Samples were shaken at 1000 rpm in a Vibrax VXR® agitator (IKA, Staufen, Germany) for 10 min and centrifuged at 1780 g in a HIMAC CF15D2 centrifuge (Hitachi, Tokyo, Japan) at 4 °C for 10 min. The organic phase was collected and evaporated in a Concentrator Plus Speed Vacuum (Eppendorf, Hamburg, Germany), and residues were solubilized in

2.3. Tebuconazole and 1-hydroxytebuconazole enantioselective analysis and method validation A Shimadzu HPLC-DAD system (section 2.2) was firstly employed in the development of the enantioselective method for the separation of TEB and TEBOH enantiomers. To this, an analytical screening strategy based on Matthijs et al. (2006) and Perrin et al. (2002) was adopted (see Supplementary Material) and the most promising conditions 97

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200 μL of methanol: water (90: 10, v/v) and submitted to LC-MS/MS analysis (section 2.3).

the Eadie-Hofstee plots, data were also fitted according to the two-site model, Eq. (3) (Hutzler et al., 2001) as follows:

2.6. In vitro kinetic parameter determination and in vitro kinetic profile after tebuconazole metabolism by HLM

v=

(Vmax1. [ S ]) + CLint 2 . [S ]2 (Km1 + [S ])

where [S] is the substrate concentration and Vmax1 and Km1 are the metabolism reaction maximum rate and the Michaelis-Menten constant for the high-affinity site, respectively, representing the pseudohyperbolic portion of the curve. CLint2 represents the slope of the linear portion of the biphasic kinetic and is defined as the low-affinity site intrinsic clearance (Vmax2/Km2 ). In turn, the high-affinity site intrinsic clearance (CLint1) was calculated as the ratio between Vmax1 and Km1 (Vmax1/Km1). Total in vitro metabolic intrinsic clearance (CLint,in vitro) was estimated by the sum of CLint1 and CLint2 (Born et al., 2000) and was scaled to in vivo intrinsic clearance (CLint,in vivo) using as scaling factor of 40 mg microsomal protein/g liver and 21.4 g liver/kg bodyweight. In vitro-in vivo extrapolations were made (Carrão et al., 2019), and hepatic clearance (CLH) and hepatic extraction ratio (EH) were calculated according to the following Eq. (4) and (5) (Bowman and Benet, 2018) (Damre and Iyer, 2012), respectively:

The in vitro kinetic parameters and the in vitro kinetic profile of TEB 1-hydroxylation catalyzed by human liver CYP450 enzymes were determined by separately incubating rac-TEB, (−)-TEB, and (+)-TEB at concentrations ranging from 0.35 to 75 μmol L−1, with HLM protein concentrations at 0.2 mg mL−1 at 37 °C for 20 min. Under these conditions, reaction rates for TEBOH enantiomer formation varied linearly with time and HLM protein concentration. These initial rate conditions (v0) were established by fixing the HLM protein concentration at 0.2 mg mL−1, varying incubation time from 0 to 50 min, and using three different rac-TEB concentrations (1, 50, and 75 μmol L−1). After incubation, reactions were stopped by addition of ethyl acetate, were subjected to the sample preparation procedure and were analyzed by the validated LC-MS/MS enantioselective analytical method (section 2.3). TEBOH enantiomers formed during the reaction were quantified using an analytical curve prepared on the same day. Then, enzymatic reaction rates of TEB 1-hydroxylation (v) were calculated by normalizing concentrations of TEBOH enantiomers formed during the reaction by microsomal protein content and incubation time. The results were plotted in graphs of v versus substrate concentration and v versus v/[S] (Eadie-Hofstee plot) using GraphPad Prism 5 software (San Diego, CA, USA). Data were analyzed as described in section 2.8.

CLH =

EH =

rac-TEB, (−)-TEB, and (+)-TEB microsomal and plasma protein binding were determined in two independent experiments by incubating the compounds (concentrations approximately 0.50 μmol L−1) with HLM (0.2 mg mL−1) or human plasma (plasma protein content of 42 mg mL−1) (Chang et al., 2010) and phosphate buffer (100 mmol L−1, pH 7.4) at 37 °C for 20 min without addition of NADPH regenerating system. Control samples, without HLM or human plasma, were prepared simultaneously. A 3000-μL aliquot of each sample was transferred to centrifuge tubes (polycarbonate, 13 × 64 mm, Beckman Coulter, Inc., Fullerton, CA, USA), and samples were ultracentrifuged at 150,000 g at 4 °C for 120 min (Optima MAX Ultracentrifuge, Beckman Coulter, Inc.). After centrifugation, 500 μL of microsome sample supernatants and 500 μL of the upper part of the plasma sample supernatant middle layer were collected, submitted to sample preparation procedures and analyzed by the LC-MS/MS method (section 2.3). The microsome unbounded fraction (fu, mic) and the plasma unbounded fraction (fu,plasm) were determined according to the following Eq. (1) (Chang et al., 2010):

(fu, plasm / fu, mic ). CLint , invivo Q + (fu, plasm / fu, mic ). CLint , invivo

. 100% (5)

2.9. CYP450 reaction phenotyping CYP450 isoforms involved in TEB 1′-hydroxylation were determined using (i) chemical inhibitors and (ii) recombinant human CYP450 isoforms (rCYPs) (Supersomes®). rac-TEB (0.50 and 50 μmol L−1), (−)-TEB (0.50 and 50 μmol L−1), and (+)-TEB (0.50 μmol L−1) were individually incubated (n = 3) at 37 °C for 20 min with pooled HLM (0.2 mg mL−1), phosphate buffer (100 mmol L−1, pH 7.4), and NADPH regenerating system in the presence of each of the following chemical inhibitors: quinidine (2 μmol L−1) for CYP2D6 (Barth et al., 2015), sulfaphenazole (10 μmol L−1) for CYP2C9 (Wustrow et al., 2012), montelukast (1 μmol L−1) for CYP2C8 (Nirogi et al., 2015), α-naphthoflavone (1 μmol L−1) for CYP1A2 (Barth et al., 2015), ketoconazole (0.50 μmol L−1) for CYP3A4/5 (Nirogi et al., 2015), ticlopidine (3 and 10 μmol L−1) for CYP2B6 and CYP2C19 (Walsky and Obach, 2007), (Barth et al., 2015), respectively, and diethyldithiocarbamate (100 μmol L−1) for CYP2E1 (Wustrow et al., 2012). For CYP2B6, ticlopidine was preincubated for 10 min with HLM and NADPH regenerating system. After that, a 10-fold dilution was made, and the reaction was initiated by TEB addition. For CYP2E1, on the other hand, diethyldithiocarbamate was preincubated for 15 min before the reaction was initiated by addition of substrates. Negative controls, in the absence of chemical inhibitors, were also prepared. The TEBOH enantiomer formation was monitored by LC-MS/MS (section 2.3), and results are expressed as percent of inhibition using the following Eq. (6). The isoforms that had their activities inhibited by at least 40% were considered to have significant participation in TEBOH enantiomer

(1)

2.8. Metabolic data analysis and in vivo toxicokinetic parameter prediction Enzyme kinetic study results were initially evaluated using Michaelis-Menten (Eq. (2)) and Eadie-Hofstee plots (v versus v/[S]).

Vmax . [ S ] Km + [S ]

(4)

where E is the hepatic extraction rate, fu, plasm is the unbounded fraction of the substrate to the plasmatic proteins, fu, mic is the unbounded fraction of the substrate to the microsomal proteins, CLint,in vivo is the in vivo intrinsic clearance and Q is the hepatic blood flow (20 mL min−1 kg−1) (Damre and Iyer, 2012).

where Csample is the TEB concentration in the samples and Ccontrol is the TEB concentration in the control.

v=

Q . (fu, plasm / fu, mic ). CLint , in vivo Q + (fu, plasm / fu, mic ). CLint , invivo

where CLH is the hepatic clearance, fu, plasm is the unbounded fraction of the substrate to the plasmatic proteins, fu, mic is the unbounded fraction of the substrate to the microsomal proteins, CLint,in vivo is the in vivo intrinsic clearance and Q is the hepatic blood flow (20 mL min−1 kg−1) (Bowman and Benet, 2018).

2.7. Tebuconazole protein binding

fu, mic or fu, plasm = Csample / Ccontrol

(3)

(2)

where Vmax and Km are the metabolism reaction maximum rate and the Michaelis-Menten constant and [S] is the substrate concentration. Since not all TEB 1-hydroxylation kinetic data followed the Michaelis-Menten model but also showed biphasic profiles according to 98

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formation (Jing et al., 2013).

%I = ((A0 − Ai )/ A0 )) x100

(6)

where A0 is the ratio between the TEBOH enantiomer and IS peak areas in negative control samples and Ai is the ratio between TEBOH enantiomer and IS peak areas in samples incubated with a chemical inhibitor. Recombinant human isoforms were also used to evaluate the rCYP1A2, rCYP2B6, rCYP2C19, rCYP2C8, rCYP2C9, rCYP2D6, rCYP2E1, rCYP3A4, and rCYP3A5 contributions (n = 3). rCYP3A5 in this case was evaluated to differentiate its contribution from rCYP3A4. Incubation mixtures contained 50 pmol mL−1 of human rCYP450 (Carrão et al., 2019), rac-TEB (0.50 or 50 μmol L−1), (−)-TEB (0.50 or 50 μmol L−1), or (+)-TEB (0.50 μmol L−1), phosphate buffer (100 mmol L−1, pH 7.4), and NADPH regenerating system. Incubation conditions were the same as the conditions used for HLM and chemical inhibitors. Control samples, composed of insect cells instead of rCYP450 isoforms, were analyzed simultaneously for comparison purposes. Analytical curves were prepared, and TEBOH enantiomer formation rates were calculated. Because rCYPs exhibit very high catalytic activities compared to their levels in HLM, the results were normalized by considering the isoform abundance in native HLM to assess the relative contribution of each CYP to TEB metabolism (Ogilvie et al., 2008). Then, the reaction rate was multiplied by the mean specific content of each CYP450 in native HLMs (Ogilvie et al., 2008) to produce a normalized rate (NR) (Rodrigues, 1999). NRs for all evaluated CYPs were summed, which gave a total normalized rate (TNR). Finally, the NR of each CYP was expressed as a percentage of TNR according to Eq. (7) as follows:

%TNR = (NR . 100)/TNR

(7)

For both methodologies, the results were plotted with the aid of GraphPad Prism 5 software (San Diego, CA, USA). Fig. 1. Representative LC-MS/MS chromatograms for enantioselective analysis of TEB samples after 20 min of metabolism by HLM (0.2 mg mL−1). A) IS (fenamiphos), B) (+)-TEBOH (retention time 8.50 min) and (−)-TEBOH (retention time 10.36 min) and C) (+)-TEB (retention time 10.27 min) and (−)-TEB (retention time 13.69 min). Blank chromatograms are highlighted, proving the selectivity of the method.

3. Results and discussion 3.1. Tebuconazole and 1-hydroxytebuconazole enantioselective analysis and elution order determination After the screening procedure to develop the enantioselective method (see Supplementary Material), the appropriated conditions for separating TEB and TEBOH enantiomers using LC-MS/MS were methanol: water (90:10, v/v) as the mobile phase at a flow rate of 0.6 mL min−1 and Chiralcel OJ® column with temperature kept at 25 °C. Initially, TEB and TEBOH enantiomers were labeled as E1 TEB (first TEB enantiomer to elute), E2 TEB (second TEB enantiomer to elute), E1 TEBOH (first TEBOH enantiomer to elute), and E2 TEBOH (second TEBOH enantiomer to elute), respectively. The optical rotation of TEB and TEBOH enantiomers was determined using CD afterwards. Samples were solubilized in the same mobile phase used during LC-MS/MS analysis, and according to the results, E1 TEB and E1 TEBOH were (+)-TEB and (+)-TEBOH, respectively (Fig. S2C and Fig. S2A). They had positive Cotton effects at 200 and 220 nm. E2 TEB and E2 TEBOH corresponded to (−)-TEB and (−)-TEBOH, respectively (Fig. S2D and Fig. S2B). They had negative Cotton effects at 200 and 220 nm. Finally, the order in which TEB and TEBOH enantiomers eluted in the developed enantioselective method was determined by individual injections of the identified enantiomers. The elution order was IS (fenamiphos, retention time at 6.47 min), (+)-TEBOH (retention time of 8.50 min), (+)-TEB (retention time of 10.27 min), (−)-TEBOH (retention time of 10.36 min), and (−)-TEB (retention time of 13.69 min) (Fig. 1). This is the first LC-MS/MS method reported for enantioselective TEB and TEBOH analysis, simultaneously.

3.2. In vitro enantioselective tebuconazole metabolism by HLM: kinetic parameters and profile determination People are constantly exposed to rac-TEB and its enantiomers through diet (Freeman et al., 2016; Saitta et al., 2017), environmental exposure (Cotton et al., 2016; Herrero-Hernández et al., 2013), and occupational exposure during its application (Fustinoni et al., 2014), which is a risk for human health. The behavior of TEB enantiomers in human body is still unknown and to help understand it, this paper evaluated the in vitro enantioselective TEB metabolism by HLM by monitoring enantioselective production of the main hydroxylated metabolite, TEBOH. Metabolism results showed that TEBOH was produced when TEB was incubated with HLM only in the presence of the co-factor NADPH. Control samples, without NADPH, did not produced TEBOH. Thus, the reaction was NADPH-dependent and catalyzed by the CYP450 enzymes present in HLM. Formation of carboxy-tebuconazole, another metabolite that has already been detected in human urine samples (Mercadante et al., 2014), was not observed in the evaluated conditions. In addition, rac-TEB incubation with HLM and NADPH produced both TEBOH enantiomers, (+)-TEBOH and (−)-TEBOH, whereas isolated (−)-TEB incubation produced only (−)-TEBOH, and isolated (+)-TEB incubation produced only (+)-TEBOH. Based on this metabolic pathway, reactions had their in vitro kinetic parameters and kinetic profiles 99

Ecotoxicology and Environmental Safety 181 (2019) 96–105

Biphasic Biphasic Biphasic Michaelis-Menten Biphasic

determined (Table 1). Isolated (+)-TEB 1-hydroxylation to give (+)-TEBOH was the only reaction that presented a typical Michaelis-Menten kinetic profile, as confirmed by the Eadie-Hofstee plot. Saturable velocities were achieved when (+)-TEB was incubated at high concentrations (Fig. 2A). In contrast, rac-TEB 1-hydroxylation producing (+/−)-TEBOH mixture, isolated (−)-TEBOH and (+)-TEBOH (Fig. 2B), and (−)-TEB 1-hydroxylation producing (−)-TEBOH (Fig. 2C), exhibited biphasic kinetic profiles without saturation even at high TEB concentrations. In these cases, reaction rates continued to increase linearly as a function of TEB concentration, which is typical of biphasic kinetics (Seibert and Tracy, 2014). Eadie-Hofstee plots clearly confirmed this atypical kinetic profile. Biphasic kinetic profiles usually occur when the reaction is catalyzed by more than one enzyme or by an enzyme with multiple binding sites (Seibert and Tracy, 2014). This profile is only observed if Km and Vmax values for multiple enzymes or multiple binding sites are sufficiently different (Seibert and Tracy, 2014). If they are similar, data will fit Michaelis-Menten kinetics (Seibert and Tracy, 2014). Kinetic parameters data were fitted to the correct models and Vmax and Km and CLint1 and CLint2 were determined, and these values were used to estimate CLint, in vitro values. CLint, in vitro indicates the functional ability of the liver's CYP450 enzymes to metabolize TEB in the absence of hepatic blood flow and protein binding. The results showed that CLint, in vitro for the rac-TEB → (−)-TEBOH reaction was approximately 1.8-fold higher than CLint, in vitro for rac-TEB → (+)-TEBOH (Table 1). So, (−)-TEB, in the absence of hepatic blood flow and protein binding, was preferentially metabolized in vitro when rac-TEB was incubated with HLM, producing a (−)-TEBOH enantiomerically enriched mixture. Therefore, when TEB is employed as a racemic mixture, both enantiomers may interact, which contributes to preferential in vitro metabolism of one enantiomer compared to the other. Shen et al. (2012) observed the same effect when they studied enantioselective TEB metabolism by rat liver microsomes. They concluded that competitive inhibition between both TEB enantiomers (Shen et al., 2012) was the reason for this difference. This competitive inhibition may also occur in in vitro incubations with HLM. In contrast, when (−)-TEB and (+)-TEB were individually incubated with HLM, despite the kinetic profiles obtained were different, CLint, in vitro values for the formation of both TEBOH enantiomers were similar (53 mL min−1 mg−1, respectively), indicating that, in the absence of hepatic blood flow and protein binding, the capacity of the liver to clear the isolated enantiomers of TEB was not different (Table 1). Furthermore, comparison between the CLint, in vitro values obtained when rac-TEB was used as substrate with the CLint, in vitro values obtained when the isolated (−)-TEB and (+)-TEB were used as substrates demonstrated that (−)-TEBOH and (+)-TEBOH were extensively produced when the isolated enantiomers were incubated as single enantiomers compared to the TEB racemic mixture. These differences in TEB metabolism would not have been verified if the analyses had been conducted by monitoring TEBOH as an achiral compound. The CLint, in vitro values for the metabolism of isolated enantiomers were similar to the CLint, in vitro value for the metabolism of rac-TEB producing (+/−)-TEBOH mixture (Table 1), which reinforced the importance of developing an enantioselective analytical method to analyze TEB and TEBOH enantiomers.

5 2 3 3 4 ± ± ± ± ± ± 0.03

52 19 33 53 53 ± 0.02 ± 0.01 ± 0.01

3.3. Toxicokinetic parameters and in vitro-in vivo (IVIVE) correlations The rac-TEB, (−)-TEB, and (+)-TEB unbounded microsomal and plasma protein fractions (fu, mic and fu, plasma) were individually determined to extrapolate in vitro data and hence obtain in vivo toxicokinetic parameters. Nonspecific microsomal binding of a substrate in in vitro kinetic studies can lead to Km overestimation, while plasma protein binding reduces the free substrate concentration that is actually available for metabolism (Wang and Gibson, 2014). Therefore, both

d

c

b

a

Data expressed as the mean ± standard error. CLint1 = intrinsic clearance (Vmax1/Km1) of the high affinity site. CLint2 = intrinsic clearance of the low affinity site (slope of the linear portion of the biphasic kinetic). CLint, in vitro = total in vitro intrinsic clearance (CLint1 + CLint2).

0.47 0.19 0.28 – 0.35 0.73 ± 0.07 1.1 ± 0.1 0.57 ± 0.06 1.0 ± 0.1 0.86 ± 0.06 Rac-TEB → (+/−)-TEBOH mixture Rac-TEB → (+)-TEBOH Rac-TEB → (−)-TEBOH (+)-TEB → (+)-TEBOH (−)-TEB → (−)-TEBOH

0.0376 0.0193 0.0190 0.0537 0.0455

± ± ± ± ±

0.0008 0.0004 0.0005 0.0005 0.0007

51 18 33 53 53

± ± ± ± ±

5 2 3 3 4

(mL min−1 mg−1) b

CLint1 Vmax1 (nmol min−1 mg−1) (μmol L−1) a

Km1 Metabolic Reaction

Table 1 Apparent kinetic parameters and profile of the enantioselective 1-hydroxylation of tebuconazole by human liver microsomes.

CLint2

c

(mL min−1 mg−1)

CLint,

in vitro

d

(mL min−1 mg−1)

Kinetic profile

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Fig. 2. Enzyme kinetic plots for the in vitro TEB 1-hydroxylation after incubation for 20 min with HLM (0.2 mg mL−1). A) (+)-TEB producing (●) (+)-TEBOH; B) rac-TEB producing (□) (+/−)-TEBOH mixture, (▲) (−)-TEBOH and (●) (+)-TEBOH; C) (−)-TEB producing (▲) (−)-TEBOH. The inserts show the Eadie-Hofstee plots. Each point is reported as the mean ± SD of triplicates.

With CLin, in vitro and unbounded fractions determined and considering hepatic blood flow (Q) of 20 mL min−1 kg−1, the enantioselective hepatic clearance (CLH) and extraction ratio (EH) of racTEB and its isolated enantiomers were calculated to predict TEB elimination from the human body due to liver metabolism (Table 2). Since the CLint, in vitro, fu, plasm, and fu, mic values of rac-TEB, (−)-TEB, and (+)-TEB differed, extrapolations indicated that CLH and EH values

parameters affect the predicted hepatic clearance accuracy and must be considered when extrapolations are conducted. The results obtained using the ultracentrifugation method showed that (−)-TEB had higher affinity for plasmatic and microsomal proteins than (+)-TEB. fu, mic and fu, plasm were 0.90 ± 0.03 and 0.06 ± 0.01 for rac-TEB, 0.95 ± 0.02 and 0.14 ± 0.03 for (+)-TEB, and 0.83 ± 0.05 and 0.01 ± 0.00 for (−)-TEB, respectively. 101

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by several isoforms, CYP3A4/5, 2E1, 2C9, 2C8, and 2C19, with greater contributions from CYP3A4/5 and CYP2E1. However, when rac-TEB was incubated at 50 μmol L−1, only CYP2E1 seemed to be involved in metabolism, with minor contribution from CYP3A4/5. In contrast, when recombinant human isoforms were employed, rCYP2C9 and rCYP3A4 were the major recombinant isoforms capable of producing (+)-TEBOH when rac-TEB was incubated at 0.50 μmol L−1, whereas the contribution of rCYP3A4 was the most significant when rac-TEB was incubated at 50 μmol L−1 (Fig. 3A). Similar results were achieved for (+)-TEBOH formation when (+)-TEB was incubated at a single concentration (0.50 μmol L−1) (Fig. 3B). (+)-TEB incubation with HLM and chemical inhibitors revealed significant contributions from CYP2C9, CYP3A4/5, and CYP2E1 in metabolism and, once again, rCYP3A4 and rCYP2C9 were the most significant recombinant isoforms to catalyze (+)-TEBOH formation. For rac-TEB 1-hydroxylation to (−)-TEBOH, the results obtained by employing chemical inhibitors showed that CYP2C8, 2E1, and 3A4/5 were involved in metabolism when the concentration was 0.50 μmol L−1, while at a high substrate concentration, CYP2E1 and CYP3A4/5 were the only enzymes involved in (−)-TEBOH formation (Fig. 3C). In addition, rCYP3A4 produced (−)-TEBOH when rac-TEB was incubated at low and high concentrations, with minor production for other rCYPs. Similar results were obtained when (−)-TEB 1-hydroxylation to (−)-TEBOH was studied (Fig. 3D). Results achieved by employing chemical inhibitors and recombinant human isoforms differed, mainly with respect to the contributions of CYP2E1 and CYP2C8. These differences could be explained by the limitations of each method and the difficulties in extrapolating the results obtained with rCYPs to the results obtained in native HLM (Ogilvie et al., 2008). Considering the method that used chemical inhibitors, CYP2C8 and CYP3A4/5 are isoforms that have active site cavities with similar sizes (Aquilante et al., 2013). Due to this size similarity, CYP2C8 and CYP3A4/5 often have overlapping substrates in HLM, which could explain why CYP2C8 was inhibited (it was involved in TEB metabolism) in HLM, but rCYP2C8 did not produce or produced low quantities of TEBOH (Jing et al., 2013). Furthermore, the chemical inhibitor used to evaluate the CYP2E1 contribution was diethyldithiocarbamate, a mechanism-based inhibitor commonly used in phenotyping reaction studies. However, diethyldithiocarbamate is already known as an inhibitor of other isoforms besides CYP2E1, such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 3A3 and 3A4 (Chang et al., 1994). This inhibition would explain why diethyldithiocarbamate inhibited rac-TEB, (+)-TEB and (−)-TEB metabolism but rCYP2E1 did not metabolize TEB to any extent (Jaakkola et al., 2006). Diethyldithiocarbamate could be inhibiting the activity of CYP3A4, the most abundant CYP isoform expressed in the human liver and an isoform that, according to the results confirmed by incubation with rCYP, contributes extensively to TEB metabolism. Other limitations may have contributed to these differences. Lipid membrane composition and protein-protein interactions taking place in HLM are different from the interactions occurring in rCYP. Membrane composition and protein-protein interactions impact enzymatic activity, resulting in different parameters even when the same enzyme and the same substrate are studied in different models. In addition, in HLM, multiple enzymes interact among themselves and with the substrate, while in rCYP, only one enzyme is overexpressed (Wang and Gibson, 2014). In conclusion, despite differences between the data obtained for each model, it can be assumed that distinct isoforms can mediate TEB metabolism, and an isoform that catalyzes production of one TEBOH enantiomer may not catalyze production of the other enantiomer or may catalyze it to a lesser extent depending on the available TEB concentration. The study with rCYP also confirmed that CYP3A4, and not CYP3A5, is the CYP3A family isoform that participates in TEB metabolism. Furthermore, it is worth pointing out that, using both models, CYP2C9 and CYP3A4 participate in (+)-TEBOH production when rac-TEB and (+)-TEB are the substrates and that CYP3A4

Table 2 Predicted enantioselective in vivo toxicokinetic parameters of tebuconazole. Metabolic Reaction

CLH (mL min−1 kg−1)

EH (%)

Rac-TEB → (+/−)-TEBOH mixture Rac-TEB → (+)-TEBOH Rac-TEB → (−)-TEBOH (+)-TEB → (+)-TEBOH (−)-TEB → (−)-TEBOH

2.6 1.0 1.7 5.0 0.53

13 5.0 8.6 25 2.7

were different for the substrates. CLH values indicated that TEB may be enantioselectively metabolized by the liver. rac-TEB metabolism producing (−)-TEBOH may occur more quickly than rac-TEB metabolism producing (+)-TEBOH (CLH ratio equal to 1.7), so a (−)-TEBOH enantiomerically enriched mixture is produced. However, when CLH values were compared for the hydroxylation of the isolated enantiomers, a preferential metabolism of (+)-TEB producing (+)-TEBOH was predicted (CLH ratio of 9.4). Concerning EH values, the human liver may be capable of poorly metabolizing TEB in its first passage. (+)-TEB seems to be easier metabolized than rac-TEB and (−)-TEB. Therefore, the (+)-TEB fraction that exits the liver unmetabolized, considering oral ingestion, and reaches systemic circulation after its first passage would be lower than rac-TEB and (−)-TEB concentrations. Consequently, the concentration of (+)-TEBOH that exits the liver would be higher than (−)-TEBOH, which should also be taken into account because there are no studies evaluating the enantioselective toxicity of TEBOH. Furthermore, although enantioselective accumulation of TEB in the human body depends on several processes, including ingestion, absorption, transportation and finally biotransformation, our study suggests that if a person ingests xenobiotics, such as drugs, that can inhibit TEB metabolism by CYP450 enzymes, or if a person suffers damage to the liver that impairs its metabolic functions, TEB could accumulate enantioselectively in the human body. This process could cause adverse effects depending on exposure levels and the enantioselective effects of the specific enantiomer on human health. Furthermore, enantioselective studies have shown that (−)-TEB degraded more slowly than (+)-TEB in aerobic and anaerobic soils (Li et al., 2015) and vegetables (Wang et al., 2012) and is preferentially accumulated in zebrafish (Liu et al., 2016). This enantioselective dissipation may result in higher human exposure to (−)-TEB than (+)-TEB, with a possible preferential accumulation of (−)-TEB in the human body. Thus, the importance of evaluating the risk of TEB to human health at the enantiomeric level is evident. 3.4. CYP450 reaction phenotyping Reaction phenotyping studies are important for identifying enzymes involved in xenobiotic metabolism. If a single CYP450 isoform mediates xenobiotic clearance from the human body, inhibition of its activity can lead to larger exposure to this compound and may cause adverse effects (Ogilvie et al., 2008). The main CYP450 isoforms involved in TEB 1-hydroxylation were identified using two TEB concentrations (0.50 and 50 μmol L−1) for the reactions with biphasic kinetic profiles (rac-TEB → (+)-TEBOH; racTEB → (−)-TEBOH; and (−)-TEB→ (−)-TEBOH) and one TEB concentration level (0.50 μmol L−1) for the reaction with the MichaelisMenten profile ((+)-TEB→ (+)-TEBOH) (Rodrigues, 1999). Two different methods were employed. First, TEB was incubated with HLM in the presence of chemical inhibitors of the major CYP450 isoforms, CYP1A2, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1, and 3A4/5. The isoforms whose activities were inhibited over 40% by the chemical inhibitors were considered involved in TEB metabolism. Then, TEB was incubated with the same rCYPs to confirm if the evaluated isoforms could catalyze production of TEBOH enantiomers. The results using HLM and chemical inhibitors showed that, at 0.50 μmol L−1 rac-TEB, (+)-TEBOH formation (Fig. 3A) was mediated 102

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Fig. 3. Determination of human CYP450 isoforms involved in the metabolism of TEB using chemical inhibitors (% Inhibition, at left) and recombinant human isoforms (%TNR, at right). The results obtained monitoring rac-TEB 1-hydroxylation producing A) (+)-TEBOH and C) (−)-TEBOH; (+)-TEB hydroxylation producing B) (+)-TEBOH; (−)-TEB hydroxylation producing D) (−)-TEBOH. Each bar is reported as the mean ± SD of triplicates.

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participates in (−)-TEBOH production when rac-TEB and (−)-TEB are the substrates. An in-silico study conducted by Jónsdótirr et al. using a quantitative structure-activity relationship QSAR model predicted that TEB was a CYP3A4 substrate in humans (Jónsdóttir et al., 2016), but it did not predict the contribution of other isoforms, as shown by our results. Considering the low values of CLH and EH for rac-TEB, (+)-TEB and specially (−)-TEB, the contribution of many CYP450 isoforms to TEB metabolism is important from a toxicological point of view (Abass et al., 2012). If one of these isoforms is inhibited, other metabolic pathways are available for hepatic clearance of TEB, which makes TEB accumulation in the body more difficult. However, the toxicity of TEBOH enantiomers originating from these reactions should also be evaluated.

carcinogenicity. Toxicol. Sci. 58, 23–31. Bowman, C.M., Benet, L.Z., 2018. An examination of protein binding and protein-facilitated uptake relating to in vitro-in vivo extrapolation. Eur. J. Pharm. Sci. 123, 502–514. https://doi.org/10.1016/J.EJPS.2018.08.008. Carrão, D.B., dos Reis Gomes, I.C., Barbosa Junior, F., de Oliveira, A.R.M., 2019. Evaluation of the enantioselective in vitro metabolism of the chiral pesticide fipronil employing a human model: risk assessment through in vitro-in vivo correlation and prediction of toxicokinetic parameters. Food Chem. Toxicol. 123, 225–232. https:// doi.org/10.1016/J.FCT.2018.10.060. Chang, T.K., Gonzalez, F.J., Waxman, D.J., 1994. Evaluation of triacetyloleandomycin, alpha-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450. Arch. Biochem. Biophys. https://doi.org/10. 1006/abbi.1994.1259. Chang, G., Steyn, S.J., Umland, J.P., Scott, D.O., 2010. Strategic use of plasma and microsome binding to exploit in vitro clearance in early drug discovery. ACS Med. Chem. Lett. 1, 50–53. https://doi.org/10.1021/ml900012h. Clasen, B., Loro, V.L., Murussi, C.R., Tiecher, T.L., Moraes, B., Zanella, R., 2018. Bioaccumulation and oxidative stress caused by pesticides in Cyprinus carpio reared in a rice-fish system. Sci. Total Environ. 626, 737–743. https://doi.org/10.1016/j. scitotenv.2018.01.154. Cotton, J., Leroux, F., Broudin, S., Poirel, M., Corman, B., Junot, C., Ducruix, C., 2016. Development and validation of a multiresidue method for the analysis of more than 500 pesticides and drugs in water based on on-line and liquid chromatography coupled to high resolution mass spectrometry. Water Res. 104, 20–27. https://doi. org/10.1016/j.watres.2016.07.075. Damre, A.A., Iyer, K.R., 2012. The significance and determination of plasma protein binding. In: Encyclopedia of Drug Metabolism and Interactions. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 1–18. https://doi.org/10.1002/9780470921920. edm032. de Albuquerque, N.C.P., Carrão, D.B., Habenschus, M.D., de Oliveira, A.R.M., 2018. Metabolism studies of chiral pesticides: a critical review. J. Pharm. Biomed. Anal. https://doi.org/10.1016/j.jpba.2017.08.011. European Medicines Agency, 2012. Guideline on bioanalytical method validation. EMA Guidel 44, 1–23. https://doi.dx.org/EMEA/CHMP/EWP/192217/2009. Freeman, S., Kaufman-Shriqui, V., Berman, T., Varsano, R., Shahar, D.R., Manor, O., 2016. Children's diets, pesticide uptake, and implications for risk assessment: an Israeli case study. Food Chem. Toxicol. 87, 88–96. https://doi.org/10.1016/j.fct. 2015.11.009. Fustinoni, S., Mercadante, R., Polledri, E., Rubino, F.M., Mandic-Rajcevic, S., Vianello, G., Colosio, C., Moretto, A., 2014. Biological monitoring of exposure to tebuconazole in winegrowers. J. Expo. Sci. Environ. Epidemiol. 24, 643–649. https://doi.org/10. 1038/jes.2014.14. Herrero-Hernández, E., Andrades, M., Álvarez-Martín, A., Pose-Juan, E., Rodríguez-Cruz, M., Sánchez-Martín, M., 2013. Occurrence of pesticides and some of their degradation products in waters in a Spanish wine region. J. Hydrol. 486, 234–245. https:// doi.org/10.1016/j.jhydrol.2013.01.025. Hutzler, J.M., Hauer, M.J., Tracy, T.S., 2001. Dapsone activation of cyp2c9-mediated metabolism: evidence for activation of multiple substrates and a two-site model. Drug Metab. Dispos. 29, 1029–1034. Jaakkola, T., Laitila, J., Neuvonen, P.J., Backman, J.T., 2006. Pioglitazone is metabolised by CYP2C8 and CYP3A4 in vitro: potential for interactions with CYP2C8 inhibitors. Basic Clin. Pharmacol. Toxicol. 99, 44–51. https://doi.org/10.1111/j.1742-7843. 2006.pto_437.x. Jing, W.-H., Song, Y.-L., Yan, R., Wang, Y.-T., 2013. Identification of cytochrome P450 isoenzymes involved in metabolism of (+)-praeruptorin A, a calcium channel blocker, by human liver microsomes using ultra high-performance liquid chromatography coupled with tandem mass spectrometry. J. Pharm. Biomed. Anal. 77, 175–188. https://doi.org/10.1016/J.JPBA.2013.01.023. Jónsdóttir, S.Ó., Reffstrup, T.K., Petersen, A., Nielsen, E., 2016. Physicologically based toxicokinetic models of tebuconazole and application in human risk assessment. Chem. Res. Toxicol. 29, 715–734. https://doi.org/10.1021/acs.chemrestox.5b00341. Li, Y., Dong, F., Liu, X., Xu, J., Han, Y., Zheng, Y., 2015. Enantioselectivity in tebuconazole and myclobutanil non-target toxicity and degradation in soils. Chemosphere 122, 145–153. https://doi.org/10.1016/j.chemosphere.2014.11.031. Liu, N., Dong, F., Xu, J., Liu, X., Zheng, Y., 2016. Chiral bioaccumulation behavior of tebuconazole in the zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 126, 78–84. https://doi.org/10.1016/j.ecoenv.2015.12.007. Lv, X., Pan, L., Wang, J., Lu, L., Yan, W., Zhu, Y., Xu, Y., Guo, M., Zhuang, S., 2017. Effects of Triazole Fungicides on Androgenic Disruption and CYP3A4 Enzyme Activity. https://doi.org/10.1016/j.envpol.2016.11.051. Matthijs, N., Maftouh, M., Vander, Heyden Y., 2006. Screening approach for chiral separation of pharmaceuticals: IV. Polar organic solvent chromatography. J. Chromatogr. A 1111, 48–61. https://doi.org/10.1016/j.chroma.2006.01.106. Mercadante, R., Polledri, E., Scurati, S., Moretto, A., Fustinoni, S., 2014. Identification and quantification of metabolites of the fungicide tebuconazole in human urine. Chem. Res. Toxicol. 27, 1943–1949. https://doi.org/10.1021/tx500291t. Nirogi, R., Palacharla, R.C., Uthukam, V., Manoharan, A., Srikakolapu, S.R., Kalaikadhiban, I., Boggavarapu, R.K., Ponnamaneni, R.K., Ajjala, D.R., Bhyrapuneni, G., 2015. Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays. Xenobiotica 45, 95–106. https://doi.org/10.3109/00498254. 2014.945196. Ogilvie, B.W., Usuki, E., Yerino, P., 2008. In Vitro Approaches for Studying the Inhibition of Drug-Metabolizing Enzymes and Identifying the Drug-Metabolizing Enzymes Responsible for the Metabolism of Drugs (Reaction Phenotyping) with Emphasis on Cytochrome P450 249–376. https://doi.org/10.3109/9780849375941-9.

4. Conclusion A reversed-phase enantioselective LC-MS/MS method was developed and validated for analysis of TEB and TEBOH enantiomers for the first time. In addition, important information concerning the TEB risk assessment for humans, at the enantiomeric level, were provided. Michaelis-Menten and biphasic kinetic profiles were observed, and in vitro-in vivo extrapolation of the toxicokinetic parameters showed that TEB may be enantioselectively cleared by the human liver, with preferential degradation of (+)-TEB, followed by rac-TEB and (−)-TEB. EH values showed that if TEB is ingested, the first pass effect will not be significant in decreasing its concentration in blood, mainly (−)-TEB concentration. In contrast, multiple isoforms, especially CYP3A4 and CYP2C9, seemed to be able to metabolize TEB to produce TEBOH. This finding is very important for reducing the risk of TEB human intoxication caused by damage to the liver or inhibition of CYP2C9 and CYP3A4 because during TEB metabolism various metabolic pathways are available. Acknowledgements The authors are grateful to the São Paulo Research Foundation (FAPESP, Grant numbers: 2013/08166-5, 2014/50945-4, 2017/032047 and 2018/07534-4), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – INCT-DATREM Process number 465571/2014-0), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001, for financial support and for granting research fellowships. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.05.071. References Abass, K.M., 2013. From in vitro hepatic metabolic studies towards human health risk assessment: two case studies of diuron and carbosulfan. Pestic. Biochem. Physiol. 107, 258–265. https://doi.org/10.1016/j.pestbp.2013.08.003. Abass, K.M., Turpeinen, M., Rautio, A., Hakkola, J., Pelkonen, O., 2012. Metabolism of pesticides by human cytochrome P450 enzymes in vitro - a survey. In: Perveen, F. (Ed.), Insectic. Adv. Integr. Pest Manag, first ed. InTech, New York, pp. 165–194. https://doi.org/10.5772/28088. Aquilante, C.L., Niemi, M., Gong, L., Altman, R.B., Klein, T.E., 2013. PharmGKB summary: very important pharmacogene information for cytochrome P450, family 2, subfamily C, polypeptide 8. Pharmacogenetics Genom. 23, 721–728. https://doi.org/ 10.1097/FPC.0b013e3283653b27. European Food Safety Authority, 2014. Conclusion on the peer review of the pesticide risk assessment of the active substance tebuconazole. EFSA J. 12 (1), 3485. https://doi. org/10.2903/j.efsa.2014.3485. Barth, T., Habenschus, M.D., Lima Moreira, F., Ferreira, L.D.S., Lopes, N.P., Moraes de Oliveira, A.R., 2015. In vitro metabolism of the lignan (−)-grandisin, an anticancer drug candidate, by human liver microsomes. Drug Test. Anal. 7, 780–786. https:// doi.org/10.1002/dta.1743. Born, S.L., Caudill, D., Smith, B.J., Lehman-McKeeman, L.D., 2000. In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and

104

Ecotoxicology and Environmental Safety 181 (2019) 96–105

M.D. Habenschus, et al.

2016). Usepa 1–33. https://doi.org/10.1017/CBO9781107415324.004. Walsky, R.L., Obach, R.S., 2007. A comparison of 2-phenyl-2-(1-piperidinyl)propane (ppp), 1,1’,1’’-phosphinothioylidynetrisaziridine (thioTEPA), clopidogrel, and ticlopidine as selective inactivators of human cytochrome P450 2B6. Drug Metab. Dispos. 35 2053–9. https://doi.org/10.1124/dmd.107.015883. Wang, Xinquan, Wang, Xuesong, Zhang, H., Wu, C., Wang, Xiangyun, Xu, H., Wang, Xiaofu, Li, Z., 2012. Enantioselective degradation of tebuconazole in cabbage, cucumber, and soils. Chirality 24, 104–111. https://doi.org/10.1002/chir.21030. Wang, Y.-H., Gibson, C.R., 2014. Variability in human In vitro enzyme kinetics. In: Methods in Molecular Biology (Clifton, N.J.), pp. 337–362. https://doi.org/10.1007/ 978-1-62703-758-7_16. Wustrow, I., Riedel, K.D., Mikus, G., Weiss, J., 2012. In vitro identification of the cytochrome P450 isozymes involved in the N-demethylation of the active opioid metabolite nortilidine to bisnortilidine. Naunyn-Schmiedeberg’s Arch. Pharmacol. 385, 633–639. https://doi.org/10.1007/s00210-012-0737-z. Zhou, J., Zhang, J., Li, F., Liu, J., 2016. Triazole fungicide tebuconazole disrupts human placental trophoblast cell functions. J. Hazard Mater. 308, 294–302. https://doi.org/ 10.1016/j.jhazmat.2016.01.055. Zhu, W., Qiu, J., Dang, Z., Lv, C., Jia, G., Li, L., Zhou, Z., 2007. Stereoselective degradation kinetics of tebuconazole in rabbits. Chirality 19, 141–147. https://doi.org/ 10.1002/chir.20340.

Perrin, C., Matthijs, N., Mangelings, D., Granier-Loyaux, C., Maftouh, M., Massart, D.L., Vander Heyden, Y., 2002. Screening approach for chiral separation of pharmaceuticals: Part II. Reversed-phase liquid chromatography. J. Chromatogr. A 966, 119–134. https://doi.org/10.1016/S0021-9673(02)00746-X. Punt, A., Peijnenburg, A.A.C.M., Hoogenboom, R.L.A.P., Bouwmeester, H., 2017. Nonanimal approaches for toxicokinetics in risk evaluations of food chemicals. ALTEX. https://doi.org/10.14573/altex.1702211. Rodrigues, A.D., 1999. Integrated cytochrome P450 reaction phenotyping. Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem. Pharmacol. https://doi.org/10.1016/S0006-2952(98) 00268-8. Saitta, M., Di Bella, G., Fede, M.R., Lo Turco, V., Potortì, A.G., Rando, R., Russo, M.T., Dugo, G., 2017. Gas chromatography-tandem mass spectrometry multi-residual analysis of contaminants in Italian honey samples. Food Addit. Contam. A 1–9. https://doi.org/10.1080/19440049.2017.1292054. Seibert, E., Tracy, T.S., 2014. Different enzyme kinetic models. In: Methods in Molecular Biology. Clifton, N.J., pp. 23–35. https://doi.org/10.1007/978-1-62703-758-7_3. Shen, Z., Zhu, W., Liu, D., Xu, X., Zhang, P., Zhou, Z., 2012. Stereoselective degradation of tebuconazole in rat liver microsomes. Chirality 24, 67–71. https://doi.org/10.1002/ chir.21027. USEPA, 2016. Chemicals Evaluated for Carcinogenic Potential (Annual Cancer Report

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