In vitro phase I metabolism of vinclozolin by human liver microsomes

Article abstract of DOI:10.1080/00498254.2018.1523485

1.?Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites. There is no information about Vin biotransformation in humans, therefore, the aim of this study was to characterize its in vitro metabolism using human liver microsomes. 2.?Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate inhibition kinetics. The K_{M app} for co-eluted M4/DTMBA and M7 was 24.2 ± 5.6 and 116.0 ± 52.6 μM, the V_{Max app} was 0.280 ± 0.015 and 0.180 ± 0.060?nmoles/min/mg protein, and the CL_{int app} was 11.5 and 1.5 mL/min/g protein, respectively. The K_i for M7 was 133.2 ± 63.9 μM. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2), ketoconazole (CYP3A4), pilocarpine (CYP2A6) and sulfaphenazole (CYP2C9) inhibited M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP. 3.?DTMBA is a stable metabolite and specific of Vin, therefore, it could be used as a biomarker of Vin exposure in humans to perform epidemiological studies.

Full text of DOI:10.1080/00498254.2018.1523485

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
In vitro phase I metabolism of vinclozolin by
human liver microsomes
Marycarmen Cruz-Hurtado, Ma. de Lourdes López-González, Victor
Mondragón & Adolfo Sierra-Santoyo
To cite this article: Marycarmen Cruz-Hurtado, Ma. de Lourdes López-González, Victor
Mondragón & Adolfo Sierra-Santoyo (2018): In vitro phase I metabolism of vinclozolin by human
liver microsomes, Xenobiotica, DOI: 10.1080/00498254.2018.1523485
To link to this article: https://doi.org/10.1080/00498254.2018.1523485
Accepted author version posted online: 14
Sep 2018.
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In vitro phase I metabolism of vinclozolin by human liver microsomes
1
1
2
Marycarmen Cruz-Hurtado, Ma. de Lourdes López-González, Victor Mondragón, and Adolfo
1
Sierra-Santoyo *
1
Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional (Cinvestav), Av. IPN No. 2508, Col. San Pedro Zacatenco, Ciudad de
2
México, CP 07360, México. Centro de Excelencia de Agilent Technologies México, Av.
Insurgentes Sur No. 1602, Oficina 801. Col. Crédito Constructor, Ciudad de México, C.P. 03940,
México.
*
Corresponding author: Adolfo Sierra-Santoyo, Centro de Investigación y de Estudios
Avanzados del IPN (Cinvestav-IPN), Departamento de Toxicología, Av. IPN No. 2508, Col. San
Pedro Zacatenco, Ciudad de México, CP 07360, México. Telephone: (52) 55 57473800, ext.
5
425. Fax: (52) 55 57473395. E-mail: asierra@cinvestav.mx

Abstract
1. Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an
endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-
carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-
methylbut-3-enanilide (M2) metabolites. There is no information about Vin
biotransformation in humans, therefore, the aim of this study was to characterize its in
vitro metabolism using human liver microsomes.
2
. Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-
oxazolidine-2,4-dione]
(M4)
and
N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-
dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually
converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly
denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-
elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate
inhibition kinetics. The KM app for co-eluted M4/DTMBA and M7 was 24.2±5.6 and
1
16.0±52.6 M, the VMax app was 0.280±0.015 and 0.180±0.060 nmoles/min/mg protein,
and the CLint app was 11.5 and 1.5 mL/min/g protein, respectively. The K for M7 was
i
1
33.2±63.9 M. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2),
ketoconazole (CYP3A4), pilocarpine (CYP2A6), and sulfaphenazole (CYP2C9) inhibited
M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP.
. DTMBA is a stable metabolite and specific of Vin, therefore it could be used as a
biomarker of Vin exposure in humans to perform epidemiological studies.
3
Key words: Vinclozolin; cytochrome P450; antiandrogen; biomarkers; xenobiotic metabolism.

Introduction
Pesticides are a heterogeneous group of chemicals to control and repel pests in different fields.
Vinclozolin (Vin) is a nonsystemic dicarboximide fungicide used to prevent rotting of several
fruits, vegetable, ornamentals plants, and turf glass (Szeto et al. 1989, US EPA 2000). Human
exposure to Vin can occur via ingestion of contaminated produce food and drink or from
occupational use.
In rats, Vin is efficiently metabolized by first undergoing a non-enzymatic hydrolysis to
generate 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-
dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites (Szeto et al. 1989; Sierra-Santoyo
et al. 2004). Regarding phase I metabolism, Vin is metabolized to at least seven metabolites by
reactions catalyzed by the cytochrome P450 (CYP) 2A, 2B, and 3A subfamilies (Sierra-Santoyo
et al. 2012). Vin is metabolized to [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-
oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-
carbamic acid (M7) metabolites; these products are unstable in physiological conditions and are
finally converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly
denoted as M5 metabolite) (Fig. 1). DTMBA represents the main metabolic product of Vin
present in organs and tissues, except in the adipose tissue, and may also be formed from M2
metabolite (Sierra-Santoyo et al. 2008). Bursztyka et al (2008), proposed that M1 is hydroxylated
in its ethylene group to form a metabolite designated as M6 and converted to DTMBA via ring
closure to form M4. The 3,5-dichloroaniline (M3) metabolite formation has been reported as a
product not NADPH-dependent from M2, however, it may not represent an important metabolite
in mammals (Sierra-Santoyo et al. 2012). With respect to phase II metabolism, glucuronide- and
sulfate-DTMBA and M4 conjugates have been reported as the main Vin metabolites excreted in
urine (Bursztyka et al. 2008, Rathahao-Paris et al. 2014, Cruz-Hurtado et al. 2018).

Although Vin exhibits low acute toxicity to wildlife (US EPA, 2000), the administration
of Vin to pregnant rats at specific gestational stages has produced a significant degree of
morphological changes, feminization, and demasculinization of male offspring (Gray et al. 1994,
Monosson et al. 1999, Wolf et al. 2000), as well as behavioral and reproductive alterations in
adult males in other species (Moorman et al. 2000, Veeramachaneni et al. 2006, Nakamura et al.
2
014, Huang et al. 2015;). These anti-androgenic effects have been associated with Vin and its
metabolites M1 and M2 (Kelce et al. 1994, 1997). The androgen receptor (AR) is competitively
inhibited by Vin, M1, and M2 (Kelce et al. 1994, Wong et al. 1995) and this inhibition interferes
with androgen-dependent gene expression (Gray et al. 1994, Monosson et al. 1999, Wolf et al.
2
000, Molina-Molina et al. 2006). However, in vivo antiandrogenic effects cannot be completely
explained by the tissue dosimetry of Vin and/or its metabolites (Sierra-Santoyo et al. 2008). In
this regard, a recent in silico study has shown that isomers of DTMBA can bind to AR of
humans, rats, and zebrafish and compete with endogenous hormones, generating antiandrogenic
effects (Galli et al. 2014).
Regarding Vin toxicity in humans, there is only one report in which M3 was used as a
biomarker of Vin exposure, but no association between anti-androgenic or reproductive effects
and urinary M3 levels was reported in an occupational setting of Vin exposure (Zober et al.
1
995). The absence of an association in this study may be due to M3 is not a specific metabolite
of V since it is also produced from other dicarboximides, such as procymidone, iprodione, and
chlozolinate (Lindh et al., 2007; Wittke et al., 2001). In a pilot study conducted in a rural
province of China (Zhejiang), in which 20 non-persistent pesticides were analyzed by gas
chromatography-mass spectrometry, Vin and acetochlor cord blood levels were significantly
associated with reduced birth weight (Wickerham et al. 2012). Despite the limited information in
humans and assuming a similar in vivo dose-response relationship and a temporal association

supported by its mechanism of action, Vin effects observed on the reproductive tract of male
animals are highly plausible in humans (Kavlock and Cummings 2005). This proposal is
supported by the fact that despite Vin instability in physiological conditions, it has been
identified in blood of umbilical cords and adult people, and placenta samples (Molina-Molina et
al. 2006, Wickerham et al. 2012).
Presently, there is no available information about Vin metabolism in humans. Thus,
extrapolation of the pathway described in rats to humans seems remote because the CYP
isoforms involved in its biotransformation have differences in catalytic activities in each specie.
The lack of information about Vin pharmacokinetics in humans indicates the need for research on
the fate of Vin to be biotransformed, accumulated, and excreted in humans and to determine
effects on the male reproductive tract associated with Vin exposure. Thus, the aim of this study
was to know the phase I biotransformation of Vin by human liver microsomes (HLM).
Materials and Methods
Chemicals
Vin (99.96% purity, lot 4355400) was purchased from Chem Service Inc. (West Chester, PA,
USA). HLM (male pool of 10, H100 Lot. 1210270) were purchased from Xenotech (Lenexa, KS,
USA). M1 and M2 metabolites were isolated as described by Szeto et al. (1989). DTMBA was
isolated from urine Vin-treated rats as described by Cruz-Hurtado et al. (2018). Methanol,
acetonitrile, and water were HPLC-grade and were purchased from Burdick and Jackson, Inc
(Muskegon, MI, USA). Furafylline, quinidine, ketoconazole, sulfaphenazole, M3, pilocarpine,
and nicotinamide adenine dinucleotide (NAPDH) were obtained from Sigma-Aldrich Chemical
Co. (Milwaukee, WI, USA). The policlonal anti-human CYP3A4 and 2C9 antibodies were
purchased from Abcam PLC (Cambridge, U.K.). The polyclonal anti-human CYP1A2 and 2D6

antibodies were obtained from Chemicon International Inc. (Temecula, CA, USA). All other
chemicals were of the highest purity available.
Enzyme assays by human liver microsomes.
The in vitro metabolism assays of Vin by HLM were carried out as previously described by
Sierra-Santoyo et al. (2012), with some modifications. Briefly, Vin (50 M) and HLM were
incubated in a final volume of 1 mL of 100 mM KH PO and 5 mM MgCl buffer at pH 7.4 in a
2
4
2
shaking water bath at 37°C. The linear conditions for Vin metabolism by HLM to determine the
enzyme kinetic parameters were experimentally established at 1 mg/mL of microsomal protein, 1
mM NADPH, and an incubation time of 30 min (data not shown). Reactions were started by
NADPH addition. After the incubation time, the reactions were finalized by the addition of 5 mL
cold acetonitrile to 100 L of incubation media, then vortexed and centrifuged (1,650xg) at 4°C
for 10 min. The supernatants were dried using sodium sulfate and evaporated under a stream of
N at 45°C. Samples were stored at 4°C and reconstituted in 100 L of acetonitrile just before the
2
chromatographic analysis. Blanks at zero time, of CO-bubbled HLM, and without NADPH were
carried out using the same enzyme assay conditions and processed for the analysis of metabolites.
All samples were prepared in triplicate and repeated three times.
Inhibition enzyme assays
To determine which CYPs are involved in Vin metabolism five CYP chemical inhibitors and four
anti-human CYP antibodies were tested. Inhibition assays were tested for CYP1A2, 2A6, 2C9,
2
D6, and 3A4, with exception of CYP2E1; these isoforms are the main human liver CYPs
involved in xenobiotic metabolism. Chemical inhibitors were preincubated in similar conditions

to those established for the enzyme assay using 50 M Vin as substrate. Inhibitors were added to
the medium enzyme assay in a maximum volume of 20 L of methanol at the following
concentrations: 0.5-3 M furafylline (CYP1A2), 0.5-4 M pilocarpine (CYP2A6), 0.6-1.5 M
sulfaphenazole (CYP2C9), 0.06-0.2 M quinidine (CYP2D6), and 0.002-0.2 M ketoconazole
(CYP3A4) (Kinonen et al. 1995, Khojastech et al. 2011). Methanol concentration in the
incubation media did not affect the CYP enzyme activities.
Inhibition assays were complemented using anti-human CYP1A2, 2C9, 2D6 and 3A4
antibodies in a final volume of 0.2 mL. For each CYP evaluated, antiserum was diluted 1:25. The
control assay without antibody and assays with antibodies. All assays were done in duplicate and
repeated twice.
Chromatographic analytical method
Vin metabolism products were quantified by HPLC/DAD using chromatographic conditions
previously reported by Sierra-Santoyo et al. (2004) with some modifications. Briefly, a Zorbax
Eclipse XDB-C18 column (5 m particle size; 4.6 mm ID × 250 mm, 80 Å) (Agilent
Technologies, Little Falls, DE, USA). System solvents consisted of methanol:acetonitrile 70:30
(A) and 0.02 M monobasic sodium phosphate pH 3.3 (B). The initial solvent conditions were set
at 40% A:60% B at a flow of 0.6 mL/min. After sample injection there was a 20 min linear
gradient change to 20% A: 80% B. Finally, there was a 5 min linear gradient change of 40%
A:60% B to reestablish initial conditions. The detector wavelength was set at 212 nm and the
reference wavelength was 550 nm. These changes did not significantly modify the recovery and
precision of Vin metabolites as described previously (Sierra-Santoyo et al. 2004). Vin
metabolites were characterized by UPLC/MSD using an Agilent Model 1290 Infinity II coupled

to a MS Q-TOF model 6545 (Agilent Technologies, Santa Clara, CA). The chromatographical
and operation conditions and the mass spectrometer was operated in negative electrospray
ionization (ESI) mode as described by Cruz-Hurtado et al. (2018).
Calibration graphics of Vin, M1, M2, and DTMBA were prepared in acetonitrile.
Concentration of metabolites M4/DTMBA and M7 were estimated using the DTMBA calibration
graphic.
Data analysis
Data of M4/DTMBA were fitted to a one-enzyme Michaelis-Menten equation V=Vmax*S/K +S.
m
M7 metabolite plot of V versus S showed a substrate inhibition kinetic, data were fitted following
the equation V=Vmax*S/K +S (1+S/K ). Eadie-Hofstee transformations plots were made to
m
i
identify the behavior of the enzyme kinetics models (Venkatakrishnan et al. 2001, Wu 2011).
Apparent kinetic parameters, Km app, Vmax app, and K for the substrate inhibition kinetic were
i
calculated by nonlinear regression using GraphPad Prism ver. 4.01 (GraphPad Software Inc., San
Diego, CA, USA).
Results
Because there is no information about nonenzymatic hydrolysis of Vin in human biological
samples, the stability of the fungicide in the presence or absence of HLM was determined. In
both conditions, Vin was gradually non-enzymatically hydrolyzed, its t1/2 in the absence of
microsomal protein was 63 min, while in the presence of microsomal protein, its t1/2 decreased to
3
8.1 min. On the contrary, the concentration of the metabolites M1 and M2 increased over time,
and M2 levels remained constant after 30 min in the presence of HLM (Fig. 2).

Incubating Vin in the presence of HLM resulted in the formation of two new peaks, in
addition to M1 and M2, which eluted at 16.8 and 17.9 min, respectively. The new peaks eluted at
1
0.3 and 12.4 min, which were designated as M4/DTMBA and M7, respectively (Fig. 3B). Only
Vin and low levels of M1 and M2 were detected in the incubation media that lacked NADPH, or
were either heat- or CO-treated or at zero-time. In a similar way, as previously reported using rat
liver microsomes (Sierra-Santoyo et al. 2012), the peak at 10.3 min corresponded to the co-
elution of two metabolites. In fresh samples immediately processed, this peak showed an UV
spectrum similar to Vin in the ascending section corresponding to M4 (Fig. 3B, inset a) and other
in the descending section similar to DTMBA (Fig. 3B, inset b). M4 and DTMBA metabolites
were identified by negative ESI mass spectrometry analysis (Fig 4). The M4 metabolite consisted
-
-
of molecular ions at m/z 317.9960 [M-H] , m/z 274.0042 [M-CO ] , m/z 246.0111 [M-
2
-
-
C H O +H O] , and m/z 159.9725 [M-C H O ] . The negative ESI data of DTMBA showed
4
8
2
2
6
8
5
-
-
molecular ions at m/z 292.0156 [M] , m/z 231.9934 [M-C H O ] , and m/z 159.9725 [M-
2
5
2
-
C H O ] . Based on these spectroscopic results, these metabolites were identified as M4 and
5
10
4
DTMBA, which have a MW of 318 and 293, respectively. The metabolite M7 is an unstable
intermediate that we were not able to identify.
After the identification of Vin metabolic products, the enzymatic kinetics of M4/DTMBA
and M7 metabolites were determined using the conditions for their linear formation by incubation
for 30 min at 37°C. Results of the enzyme kinetic of M4/DTMBA metabolites showed a simple
monophasic and hyperbolic kinetic (r=0.976), which was verified by the Eadie-Hofstee plot and
fit to the Michaelis-Menten plot (Fig. 5). Results of the enzyme kinetic analysis of M7 showed a
substrate inhibition kinetic, which was verified by the Eadie-Hofstee plot. The calculated KM app
corresponding to the co-elution peak M4/DTMBA (24.2 ± 5.6 M) was lower than that calculated

for M7 (116.0 ± 52.6 M). The VMax app values for M4/DTMBA and M7 were 0.280 ± 0.015 and
.184 ± 0.060 nmoles of product/min/mg protein, respectively. The intrinsic clearance (CLint
0
app=VMax app/KM app) values for M4/DTMBA and M7 were 11.5 and 1.5 mL/min/g protein,
respectively, and the inhibitory constant (K ) calculated for M7 in the inhibitory substrate kinetic
i
was 133.2 ± 63.9 M.
To determine which were the main CYPs involved in Vin metabolism, five chemical
inhibitors for the main human liver CYPs were tested. Furafylline and ketoconazole were the
main inhibitors of the formation of M4/DTMBA and M7 metabolites. The half inhibitory
concentrations (IC ) of ketoconazole for M4/DTMBA and M7 formation were 0.280 and 0.091
5
0
M, respectively; the IC50 of furafylline for M4/DTMBA and M7 was 2.2 and 1.3 M,
respectively. Pilocarpine and sulfaphenazole strongly inhibited the M7 formation, the IC was
5
0
0
.227 and 1.247 M, respectively (Fig. 6). The addition of anti-human CYP1A2, 2C9, and 3A4
antibodies to the incubation media at the dilution assayed completly inhibited the production of
M4/DTMBA and M7 metabolites. Vin metabolism was not affected by the addition of quinidine
or anti-human CYP2D6 antibody to the incubation medium.
Preliminary studies on M1 and M2 metabolism indicate that M2 was metabolized to M5,
and trace levels of M3 were detected from 100 M of substrate. M1 was not metabolized under
the enzyme assay conditions used (results not shown).
Discussion
The main finding of this study was that phase I Vin metabolism in humans is CYP-dependent and
its metabolic pathway is similar to that described for rats (Fig. 1). Vin was non-enzymatically
hydrolyzed to M1 and M2 metabolites in the presence and absence of HLM (Fig. 2). However,

the Vin hydrolysis in the presence of microsomal protein enhanced. These results confirm that
Vin is very susceptible to hydrolysis in basic media and is favored by the presence of proteins,
lipids and ions, as has been previously reported when Vin was incubated in phosphate buffer pH
7
.4 and rat serum (Szeto et al. 1989, Sierra-Santoyo et al. 2004). Likewise, Vin is metabolized to
M4 and M7 metabolites, which are unstable and spontaneously converted to DTMBA by some
CYPs. It is possible that Vin undergoes a CYP-dependent epoxidation reaction on the vinyl group
to generate the epoxide-M4, followed by an epoxyde hydrolase reaction or by spontaneous
hydrolysis to form the M4 metabolite, and the opening of the 2,4-oxazolidinedione ring by the
addition of water resulting in the formation of M7, which undergoes decarboxylation to finally
form DTMBA. Another pathway of biotransformation is through M7, bypassing M4 and
undergoing a rearrangement to finally form DTMBA.
DTMBA was the most stable metabolite of Vin; in incubations longer than 30 min, the
disappearance of M4 and M7 was remarkable, which resulted in DTMBA as the final product.
These results are in agreement with those described in in vitro and in vivo studies in the rat, in
which DTMBA was the main Vin metabolite of phase I (Bursztyka et al. 2008, Sierra-Santoyo et
al. 2008, 2012). Likewise, DTMBA could also be generated from M2 by an enzymatic
epoxidation of the ethylene group followed by the hydrolysis of the epoxide (Sierra-Santoyo et
al. 2008). To date, we do not have evidence about the formation of the intermediary epoxide
either from Vin or M2 metabolite. In this study, the M3 metabolite was not detected as a product
of Vin biotransformation by HLM. A possible explanation could be that it is generated at very
low concentrations and could only be originated from the M2 biotransformation. This possibility
is supported by our preliminary studies detecting M3 at trace levels from 100 M of M2 as
substrate (results not shown). Currently, there is no information about M1 and M2 metabolism in

humans as yet; therefore, further studies are needed to determine the metabolic fate of M1 and
M2 in humans, and if M3 is formed from M2, as it was reported using rat liver microsomes
(RLM) (Sierra-Santoyo et al. 2012). Although not fully addressed, preliminary studies on
enzymatic hydrolysis of Vin, M1, and M2 using hepatic or serum esterases obtained from rat or
human have shown lack of enzymatic activity (results not shown). These results also proved that
M3 is a minor metabolic product and is not a specific metabolite of Vin, which indicates that it
does not meet the necessary criteria to be used as biomarker of Vin exposure.
In the current study, we demonstrated that Vin is metabolized in humans by liver
microsomal CYP-dependent monooxygenases. The results of M4/DTMBA and M7 formation
using chemical inhibitors and anti-human CYP antibodies indicated a differential effect on each
metabolite. Furafylline, a human CYP1A2 inhibitor (Ono et al. 1996), inhibited both
M4/DTMBA and M7 formation, showing IC values of 2.2 and 1.348 M, respectively. These
5
0
values are within the range of the Ki values (0.6-4.4 M) determined by phenacetin O-deethylase
activity (Bourrié et al. 1996, McKillop et al. 1998). Ketoconazole has been recognized as a
powerful inhibitor of CYP3A subfamily and other CYPs in several species including the human
(Ono et al. 1996, Khojastech et al. 2011). In this study, ketoconazole inhibited M4/DTMBA and
M7 formation, with an IC of 0.287 and 0.091 M, respectively. These values are consistent
50
with those reported for azamulin, a potent and selective inhibitor of CYP3A (Stresser et al.
004). In contrast, sulfaphenazole and pilocarpine inhibited M7 formation; sulfaphenazole is a
2
potent and very selective inhibitor of CYP2C subfamily, although CYP2C9 is the most
susceptible isoform to be affected (Ono et al. 1996, Khojastech et al. 2011). Our data showed that
sulfaphenazol had an IC of 1.247 M; this value is very close to the Ki value reported for
5
0
CYP2C9 determined by the tolbutamide 4-hydroxylase activity (Bourrié et al. 1996, Komatsu et

al. 2000). Pilocarpine has been shown to inhibit CYP2A, 2B, and 3A activities in human and
mouse (Kinonen et al. 1995). The IC of pilocarpine calculated in this study (0.227 M) is much
5
0
lower than the IC calculated for Vin as substrate using RLM (22.6 M) (Sierra-Santoyo et al.
5
0
2
012) and for 7-hydroxylation of coumarin using HLM (4 M) (Bourrié et al. 1996). Together,
these results suggest that CYP1A2, 2A6, 2C9, and 3A4 could be the main enzymes involved in
Vin biotransformation in humans. This proposal is supported by the results observed when anti-
human CYP antibodies were added to the enzymatic assay medium. These results represent the
first approach to determine CYPs involved in Vin metabolism in humans. Further studies are
needed to determine a more reliable individual contribution using recombinant heterologously-
expressed human CYPs.
This is the first report describing kinetic parameters, such as KM app, VMax app, and CLint app
of Vin biotransformation by HLM. Rane et al (1977) proposed that the in vitro CL may be used
int
to link the fundamental enzymatic kinetics (K and VMax) and in vivo pharmacokinetic variables,
M
in addition it may help to predict the rate of hepatic clearance in vivo and to define the
contribution of individual metabolic pathways when a substrate is metabolized to more than two
products (Obach 1999, Sjögren et al. 2009). In this study, the enzymatic kinetic parameters were
individually obtained despite the instability of the intermediary metabolites in a similar manner
as they were calculated for RLM (Sierra-Santoyo et al. 2012). The CLint app for M4/DTMBA was
7
.6-fold higher than that of M7, which indicates a preferential formation of M4/DTMBA from
Vin by HLM, similar to that of RLM (Sierra-Santoyo et al. 2012). This result suggests that Vin
can be easily metabolized to M4 to be finally converted to DTMBA. M7 metabolite formation
showed substrate inhibition kinetics; this behavior describes a process in which metabolic rate
decreases at high substrate levels and is verified by a low formation of product reflected by a low

value of CLint app. These results also suggest that in humans, DTMBA could represent the main
metabolite in people exposed to Vin because it is the most stable metabolite.
The results of this in vitro study using HLM indicate that M3 is not formed from Vin;
therefore, M3 is not a specific metabolic product of Vin. In several studies, M3 has been detected
in human urine samples (Wittke et al. 2001, Weiss and Angerer 2002, Turci et al. 2006, Lindh et
al. 2007, Kutting et al. 2009). The presence of M3 in these studies could be due to the acidic or
alkaline conditions used for its detection (Szeto et al. 1989, Sierra-Santoyo et al. 2004). In the
only report where M3 was used as a biomarker, there was no association between antiandrogenic
or reproductive effects and urinary M3 levels in an occupational setting of Vin exposure (Zober
et al. 1995). In contrast, in a pilot study conducted in Zhejiang, a rural province in China, Vin
and acetochlor cord blood levels were significantly associated with reduced birth weight
(Wickerham et al. 2012). Molina-Molina et al (2006) reported a high frequency of Vin in blood
samples obtained from young people living in southern Spain. Based on our results and assuming
similar pharmacokinetics of Vin in humans as it was described in rats (Sierra-Santoyo et al.
2
008), very high levels of other metabolites, mainly DTMBA, should be detected in those human
samples. Thus, DTMBA could represent an excellent alternative to be used as a biomarker of Vin
exposure because is easly detected after enzymatic hydrolysis of its conjugated metabolites
(Bursztyka et al. 2008, Rathahao-Paris et al. 2014, Cruz-Hurtado et al. 2018).
In summary, the current study is the first report describing the in vitro Vin metabolism by
HLM. Vin was metabolized in vitro to M4 and M7 products, which are chemically unstable and
by a molecular rearrangement they are transformed to DTMBA. Vin metabolism is CYP-
dependent and CYP1A2, 3A4, 2A6, and 2C9, could be the main isoforms involved in its
biotransformation. More studies about M1 and M2 metabolism are needed to complete the
metabolic pathway of Vin. DTMBA is a stable metabolite and specific of Vin, therefore it may be

used as a biomarker of exposure to Vin in pharmacokinetic and toxicological studies. There is no
available toxicological information about M4, DTMBA, or M7 metabolites; then, the
toxicological characterization of these metabolites is needed for a better understanding of male
reproductive effects in humans associated with V exposure.
Acknowledgments
Marycarmen Cruz-Hurtado was a recipient of a scholarship from Consejo Nacional de Ciencia y
Tecnología-México (Conacyt) (Number 421556). This work was supported by Conacyt (Grant
number 168384).
Conflicts of interest
The authors declare that there are no conflicts of interest.

References
Bourrie, M., Meunier, V., Berger, Y., and Fabre, G., 1996. Cytochrome P450 isoform inhibitors
as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J
Pharmacol Exp Ther 277:321-332.
Bursztyka, J., Debrauwer, L., Perdu, E., Jouanin, I., Jaeg, J.P., and Cravedi, J.P., 2008.
Biotransformation of vinclozolin in rat precision-cut liver slices: comparison with in vivo
metabolic pattern. J Agric Food Chem 56:4832-4839.
Cruz-Hurtado, M., López-González, M.L., Escobar-Wilches, D.C., and Sierra-Santoyo, A.
(2018). Analysis of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA) as a new
potential biomarker of exposure to vinclozolin in urine. Toxicol Appl Pharmacol 346:1-8.
Galli, C.L., Sensi, C., Fumagalli, A., Parravicini, C., Marinovich, M., and Eberini, I., 2014. A
computational approach to evaluate the androgenic affinity of iprodione, procymidone,
vinclozolin and their metabolites. PLoS One 9: e104822.
Gray, L.E., Jr., Ostby, J.S., and Kelce, W.R., 1994. Developmental effects of an environmental
antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol Appl
Pharmacol 129:46-52.
Huang, Y., Wang, X.L., Zhang, J.W., and Wu, K.S., 2015. Impact of endocrine-disrupting
chemicals on reproductive function in zebrafish (Danio rerio). Reprod Domest Anim 50:1-6.
Kavlock, R., and Cummings, A., 2005. Mode of action: inhibition of androgen receptor function-
-vinclozolin-induced malformations in reproductive development. Crit Rev Toxicol 35:721-
7
26.
Kelce, W.R., Lambright, C.R., Gray, L.E., Jr., and Roberts, K.P., 1997. Vinclozolin and p,p'-
DDE alter androgen-dependent gene expression: in vivo confirmation of an androgen
receptor-mediated mechanism. Toxicol Appl Pharmacol 142:192-200.

Kelce, W.R., Monosson, E., Gamcsik, M.P., Laws, S.C., and Gray, L.E., Jr., 1994.
Environmental hormone disruptors: evidence that vinclozolin developmental toxicity is
mediated by antiandrogenic metabolites. Toxicol Appl Pharmacol 126:276-285.
Khojasteh, S.C., Prabhu, S., Kenny, J.R., Halladay, J.S., and Lu, A.Y., 2011. Chemical inhibitors
of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform
selectivity. Eur J Drug Metab Pharmacokinet 36:1-16.
Kinonen, T., Pasanen, M., Gynther, J., Poso, A., Jarvinen, T., Alhava, E., and Juvonen, R.O.,
1
995. Competitive inhibition of coumarin 7-hydroxylation by pilocarpine and its interaction
with mouse CYP 2A5 and human CYP 2A6. Br J Pharmacol 116:2625-2630.
Komatsu, T., Yamazaki, H., Asahi, S., Gillam, E.M., Guengerich, F.P., Nakajima, M., and Yokoi,
T., 2000. Formation of a dihydroxy metabolite of phenytoin in human liver
microsomes/cytosol: roles of cytochromes P450 2C9, 2C19, and 3A4. Drug Metab Dispos
2
8:1361-1368.
Kutting, B., Goen, T., Schwegler, U., Fromme, H., Uter, W., Angerer, J., and Drexler, H., 2009.
Monoarylamines in the general population--a cross-sectional population-based study including
1
004 Bavarian subjects. Int J Hyg Environ Health 212:298-309.
Lindh, C.H., Littorin, M., Amilon, A., and Jonsson, B.A., 2007. Analysis of 3,5-dichloroaniline
as a biomarker of vinclozolin and iprodione in human urine using liquid chromatography/triple
quadrupole mass spectrometry. Rapid Commun Mass Spectrom 21:536-542.
McKillop, D., Wild, M.J., Butters, C.J., and Simcock, C., 1998. Effects of propofol on human
hepatic microsomal cytochrome P450 activities. Xenobiotica 28:845-853.
Molina-Molina, J.M., Hillenweck, A., Jouanin, I., Zalko, D., Cravedi, J.P., Fernandez, M.F.,
Pillon, A., Nicolas, J.C., Olea, N., and Balaguer, P., 2006. Steroid receptor profiling of
vinclozolin and its primary metabolites. Toxicol Appl Pharmacol 216:44-54.
Monosson, E., Kelce, W.R., Lambright, C., Ostby, J., and Gray, L.E., Jr., 1999. Peripubertal
exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the

development of androgen-dependent tissues, and alters androgen receptor function in the male
rat. Toxicol Ind Health 15:65-79.
Moorman, W.J., Cheever, K.L., Skaggs, S.R., Clark, J.C., Turner, T.W., Marlow, K.L., and
Schrader, S.M., 2000. Male adolescent exposure to endocrine-disrupting pesticides:
vinclozolin exposure in peripubertal rabbits. Andrologia 32:285-293.
Nakamura, A., Takanobu, H., Tamura, I., Yamamuro, M., Iguchi, T., and Tatarazako, N., 2014.
Verification of responses of Japanese medaka (Oryzias latipes) to anti-androgens, vinclozolin
and flutamide, in short-term assays. J Appl Toxicol 34:545-553.
Obach, R.S., 1999. Prediction of human clearance of twenty-nine drugs from hepatic microsomal
intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding
to microsomes. Drug Metabol Disp 27:1350-1359.
Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F.J., and Tsutsui, M., 1996. Specificity of
substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using
cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26:681-693.
Rane, A., Wilkinson, G.R., and Shand, D.G., (1977). Prediction of hepatic extraction ratio from
in vitro measurement of intrinsic clearance. J Pharmacol Exp Therap 200:420-424.
Rathahao-Paris, E., Paris, A., Bursztyka, J., Jaeg, J.P., Cravedi, J.P., and Debrauwer, L., 2014.
Identification of xenobiotic metabolites from biological fluids using flow injection analysis
high-resolution mass spectrometry and post-acquisition data filtering. Rapid Commun Mass
Spectrom 28:2713-2722.
Sierra-Santoyo, A., Angeles-Soto, E., de Lourdes Lopez-Gonzalez, M., Harrison, R.A., and
Hughes, M.F., 2012. In vitro metabolism of the anti-androgenic fungicide vinclozolin by rat
liver microsomes. Arch Toxicol 86:413-421.
Sierra-Santoyo, A., Barton, H.A., and Hughes, M.F., 2004. Liquid chromatography determination
of the anti-androgen vinclozolin and its metabolites in rat serum. J Chromatogr B Analyt
Technol Biomed Life Sci 809:105-110.

Sierra-Santoyo, A., Castaneda-Hernandez, G., Harrison, R.A., Barton, H.A., and Hughes, M.F.,
008. Pharmacokinetics and dosimetry of the antiandrogen vinclozolin after oral
2
administration in the rat. Toxicol Sci 106:55-63.
Sjögren, E., Lennernäs, H., Andersson, T.B., Gråsjö, J., and Bredberg, U., (2009). The multiple
depletion curves method provides accurate estimates of intrinsic clearance (CL ), maximum
int
velocity of the metabolic reaction (Vmax), and Michaelis constant (K ): accuracy and
m
robustness evaluated through experimental data and Monte Carlo simulations. Drug Metabol
Disp 37:47-58.
Stresser, D.M., Broudy, M.I., Ho, T., Cargill, C.E., Blanchard, A.P., Sharma, R., Dandeneau,
A.A., Goodwin, J.J., Turner, S.D., Erve, J.C., Patten, C.J., Dehal, S.S., and Crespi, C.L., 2004.
Highly selective inhibition of human CYP3Aa in vitro by azamulin and evidence that
inhibition is irreversible. Drug Metab Dispos 32:105-112.
Szeto, S.Y., Burlinson, N.E., Rahe, J.E., and Oloffs, P.C., 1989. Kinetics of hydrolysis of the
dicarboximide fungicide vinclozolin. J Agricul Food Chem 37:523-529.
Turci, R., Barisano, A., Balducci, C., Colosio, C., and Minoia, C., 2006. Determination of
dichloroanilines in human urine by gas chromatography/mass spectrometry: validation
protocol and establishment of Reference Values in a population group living in central Italy.
Rapid Commun Mass Spectrom 20:2621-2625.
US EPA, R.-P., 2000. R.E.D. FACTS Vinclozolin vol. 2013.
Veeramachaneni, D.N., Palmer, J.S., Amann, R.P., Kane, C.M., Higuchi, T.T., and Pau, K.Y.,
2
006. Disruption of sexual function, FSH secretion, and spermiogenesis in rabbits following
developmental exposure to vinclozolin, a fungicide. Reproduction 131:805-816.
Venkatakrishnan, K., Von Moltke, L.L., and Greenblatt, D.J., 2001. Human drug metabolism and
the cytochromes P450: application and relevance of in vitro models. J Clin Pharmacol
4
1:1149-1179.
Weiss, T., and Angerer, J., 2002. Simultaneous determination of various aromatic amines and
metabolites of aromatic nitro compounds in urine for low level exposure using gas

chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci
78:179-192.
7
Wickerham, E.L., Lozoff, B., Shao, J., Kaciroti, N., Xia, Y., and Meeker, J.D., 2012. Reduced
birth weight in relation to pesticide mixtures detected in cord blood of full-term infants.
Environ Int 47:80-85.
Wittke, K., Hajimiragha, H., Dunemann, L., and Begerow, J., 2001. Determination of
dichloroanilines in human urine by GC-MS, GC-MS-MS, and GC-ECD as markers of low-
level pesticide exposure. J Chromatogr B Biomed Sci Appl 755:215-228.
Wolf, C.J., LeBlanc, G.A., Ostby, J.S., and Gray, L.E.Jr., 2000. Characterization of the period of
sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci 55:152-161.
Wong, C., Kelce, W.R., Sar, M., and Wilson, E.M., 1995. Androgen receptor antagonist versus
agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem
2
70:19998-20003.
Wu, B., 2011. Substrate inhibition kinetics in drug metabolism reactions. Drug Metab Rev
3:440-456.
4
Zober, A., Hoffmann, G., Ott, M.G., Will, W., Germann, C., and van Ravenzwaay, B., 1995.
Study of morbidity of personnel with potential exposure to vinclozolin. Occup Environ Med
5
2:233-241.

Footnotes
Figure 1. Proposed metabolic pathway of Vin by human liver microsomes.
Figure 2. Non-enzymatic hydrolysis of Vin in enzyme assay in the presence or absence of human
liver microsomes. (A) depletion profile of Vin; (B) generation of the M1 metabolite; (C)
generation of the M2 metabolite. Incubations were carried out using 50 μM Vin in 200 mM
KH PO and 5 mM MgCl , pH 7.4 at 37 °C (n=3).
2
4
2
Figure 3. Typical HPLC chromatograms of: (A) standard solutions of Vin (32.5 M), M1 (16.5
M), M2 (19 M), and M3 (31 M) in acetonitrile; (B) extract of the in vitro enzyme assay of 50
M Vin by human liver microsomes. Inset includes the UV absorption spectra of metabolites M4
(a), DTMBA (b), and M7 (c). Metabolites were extracted in acetonitrile from the enzyme assay
incubation medium. Extracts were dried under a stream of N , and the residue was reconstituted
2
in 100 L of acetonitrile. See Materials and Methods section for experimental details.
Figure 4. HPLC/mass spectrometry chromatogram of metabolites generated by the
biotransformation of Vin (50 M) by human liver microsomes (A), negative ionization mass
spectra of metabolites DTMBA (B), and M4 (C). System solvents consisted of
water:acetonitrile:1% formic acid (70:30:0.1).
Figure 5. Substrate saturation plots of products generated by in vitro Vin metabolism by human
liver microsomes. (A) M4/DTMBA metabolites, and (B) M7 metabolite. Inserts depict Eadie-
Hofstee plots. Vin was incubated in 200 mM KH PO , 5 mM MgCl , human liver microsomes (1
2
4
2
mg/mL), and 1 mM NADPH, pH 7.4 at 37 °C during 30 min. Data represent the mean ± standard
deviation (n=3).
Figure 6. Effect of chemical inhibitors furafylline (A), pilocarpine (B), sulfaphenazole (C), or
ketoconazole (D) on M4/DTMBA and M7 formation from Vin by human liver microsomes. Vin
(
50 M) was incubated in 200 mM KH PO , 5 mM MgCl , human liver microsomes (1 mg/mL),
2
4
2
and 1 mM NADPH, pH 7.4 at 37 °C (n=3).