Sulphoxidation of ethyl methyl sulphide, 4-chlorophenyl methyl sulphide and diphenyl sulphide by purified pig liver flavin-containing monooxygenase

Article abstract of DOI:10.1080/0049825021000022339

1. The biotransformation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) to their corresponding sulphoxides by purified flavin-containing monooxygenase (FMO) is described. 2. Purified pig liver flavin-containing monooxygenase catalysed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides and the reactions followed single enzyme Michelis-Menten kinetics. 3. The apparent K_m and V_max for the sulphoxidation of EMS were 1.38 ± 0.05 mM and 78.74 ± 3.9 nmoles mg^-1 protein min^-1, respectively. The apparent K_m and V_max for the sulphoxidation of CPMS were 0.185 ± 0.03 mM and 103 ± 5.0 nmoles mg^-1 protein min^-1, respectively. The apparent K_m and V_max for the sulphoxidation of DPS were 0.068 ± 0.002 mM and 49.26 ± 2.05 nmoles mg^-1 protein min^-1, respectively. 4. A significant reduction of the sulphoxidation of these simple sulphides was observed with addition of 1-naphthylthiourea in the incubation medium. On the other hand, incorporation of catalase and superoxide dismutase into the incubation media produced no appreciable inhibition of the observed sulphoxidation of the sulphides. 5. These results suggest that FMO is responsible, at least in part, for the sulphoxidation of nucleophilic sulphides as well as for the oxidation of sulphur atoms that reside within or adjacent to aromatic systems.

Full text of DOI:10.1080/0049825021000022339

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Sulphoxidation of ethyl methyl sulphide, 4-
chlorophenyl methyl sulphide and diphenyl
sulphide by purified pig liver flavin-containing
monooxygenase
I. P. Nnane & L. A. Damani
To cite this article: I. P. Nnane & L. A. Damani (2003) Sulphoxidation of ethyl methyl sulphide,
4-chlorophenyl methyl sulphide and diphenyl sulphide by purified pig liver flavin-containing
monooxygenase, Xenobiotica, 33:1, 83-91
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Date: 10 November 2015, At: 15:28

xenobiotica, 2003, vol. 33, no. 1, 83–91
Sulphoxidation of ethyl methyl sulphide, 4-
chlorophenyl methyl sulphide and diphenyl
sulphide by purified pig liver flavin-containing
monooxygenase
I. P. NNANE{* and L. A. DAMANI{
{
Department of Pharmaceutical Sciences, Temple UniversitySchool of Pharmac y, 3307
N. Broad Street, Philadelphia, PA 19140, USA
Department of Pharmacy, The Chinese University of Hong Kong, Shatin, Hong Kong,
{
P. R. China
Received 19 June 2002
1. The biotransformation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl
sulphide (CPMS) and diphenyl sulphide (DPS) to their corresponding sulphoxides by
purified flavin-containing monooxygenase (FMO) is described.
2. Purified pig liver flavin-containing monooxygenase catalysed the sulphoxidation of
EMS, CPMS and DPS to their corresponding sulphoxides and the reactions followed
single enzyme Michelis–Menten kinetics.
3
. The apparent K
and 78.74 ꢀ 3.9 nmoles mg protein min , respectively. The apparent K
the sulphoxidation of CPMS were 0.185 ꢀ 0.03 mm and 103 ꢀ 5.0 nmoles mg
m
and Vmax for the sulphoxidation of DPS
m
and Vmax for the sulphoxidation of EMS were 1.38 ꢀ 0.05 mm
ꢁ1 ꢁ1
m
and Vmax for
ꢁ1
ꢁ
1
protein min , respectively. The apparent K
were 0.068 ꢀ 0.002 mm and 49.26 ꢀ 2.05 nmoles mg protein min , respectively.
ꢁ
1
ꢁ1
4. A significant reduction of the sulphoxidation of these simple sulphides was
observed with addition of 1-naphthylthiourea in the incubation medium. On the other
hand, incorporation of catalase and superoxide dismutase into the incubation media
produced no appreciable inhibition of the observed sulphoxidation of the sulphides.
5
. These results suggest that FMO is responsible, at least in part, for the
sulphoxidation of nucleophilic sulphides as well as for the oxidation of sulphur atoms
that reside within or adjacent to aromatic systems.
Introduction
Sulphide and sulphoxide functionalities are frequentlyencountered in agricul-
tural chemicals and medicinal agents and maydetermine or influence the biological
fate of the parent molecule (Ziegler 1980). Sulphides are readilyconverted to
sulphoxides in vitro and in vivo due to the readilyaccessible lone pair of electrons
on the divalent sulphur atom (Ziegler 1990). The psychoactive phenothiazine
drugs, for example, are metabolized to their corresponding sulphoxides in various
animal species and in man (Traficante et al. 1979). Sulphoxides, on the other hand,
maybe oxidized to the corresponding sulphones or reduced to sulphides. The
sulphoxide drug, sulphinpyrazone, undergoes both reduction to the corresponding
sulphide and oxidation to the sulphone after parenteral administration in rats
(
Renwick 1982).
The mammalian flavin-containing monooxygenase (FMO) is a family of
enzymes mainly distributed in the liver, lung and kidney of mammals (Ziegler
*
Author for correspondence; e-mail: ivo.nnane@temple.edu
Xenobiotica ISSN 0049–8254 print/ISSN 1366–5928 online # 2003 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/0049825021000022339

8
4
I. P. Nnane and L. A. Damani
1
988). FMOs exhibit broad substrate specificityfor catal sy ing sulphur and
nitrogen functional groups, which are common in therapeutic drugs (Ziegler
990). Additionally, FMO is a major contributor to the metabolism of agricultural
chemicals and maybe responsible for producing biologicallyactive metabolites
Ziegler 1988). To date, five distinct members of this protein family, designated
1
(
FMO1–5, have been identified in mammals including humans. FMO1, the
dominant form of FMO in pig liver, has been reported to S-oxygenate a number
of nucleophilic sulfur-containing substrates (Lawton et al. 1994). There is a
considerable amount of information available on the pig liver FMO1 compared
with the human form. However, the substrate specificityof human FMO1 appears
to be considerablymore restricted than that of the pig FMO1. Therefore,
differences in the types of nucleophiles accepted must be considered in attempting
to extrapolate the extensive structure–activitystudies available for the pig FMO1
to the human FMO1 (Kim and Ziegler 2000). Interestingly, the specific role of the
FMOs in oxidative metabolism of xenobiotics in mammals is not well defined due
to lack of specific and selective inhibitors for the FMO forms.
Ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and
diphenyl sulphide (DPS) (figure 1) are simple sulphides that are encountered in
petroleum distillates and in the environment. These compounds are also inter-
mediates in the manufacture of other xenobiotics. EMS, CPMS and DPS are
biotransformed to their corresponding sulphoxides in vitro and in vivo (Nnane and
Damani 1999, 2002). The cytochrome P450 or the FMO enzyme systems are
Figure 1. Sulphoxidation of (a) ethyl methyl sulphide (EMS), (b) 4-chlorophenyl methyl sulphide
(
CPMS) and (c) diphenyl sulphide (DPS) to their corresponding sulphoxides.

Sulphoxidation of simple sulphides
85
thought to mediate these S-oxidation reactions depending on the nucleophilicityof
the substrates (Poulsen and Ziegler 1995). It has been reported that FMO is
responsible for the sulphoxidation of the more nucleophilic aliphatic sulphides to
their corresponding sulphoxides while both cytochrome P450 and the FMO are
involved in the oxidation of sulphur atoms that reside within or adjacent to
aromatic or heterocyclic ring systems (Hoodi and Damani 1984, Nnane and
Damani 2002). Previous studies have also reported that the metabolism of a
simple dialkyl sulphide such as EMS to its sulphoxide is predominantly mediated
byFMO whereas sulphoxidation of an alk lya r ly sulphide such as CPMS is
mediated byboth c tyo chrome P450 and the FMO in rat liver microsomes
(
Nnane and Damani 1999, 2002). These studies also indicate that the sulphoxida-
tion of DPS to its sulphoxide is mediated almost exclusivelybyc yt ochrome P450
in rat liver microsomes. The aim of the present studywas to determine the specific
role of purified pig liver FMO in the sulphoxidation of these simple sulphides to
their corresponding sulphoxide metabolites. The information obtained on the
sulphoxidation of these simple sulphides bythe pig liver FMO maybe extra-
polated to S-oxidation reactions involving more complex xenobiotics bearing the
thioether functionality.
Materials and methods
Chemicals
Ethyl methyl sulphide (98%), 4-chlorophenyl methyl sulphide (98%), diphenyl sulphide (98%), n-
propyl sulphone, 1-naphthylthiourea, 4-chlorophenyl phenyl sulphone, nicotinamide adenine dinucleo-
tide (NADP ), glucose 6-phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PD) superoxide
+
ꢁ1
dismutase and catalase (11 000 units mg protein) were purchased from Sigma Chemical Co. (St Louis,
MO, USA). The sulphoxides of EMS, CPMS and DPS were synthesized in our laboratory by methods
described byMadesclaire (1986). The purityof each sulphoxide was >98%. All other reagents and
organic solvents of HPLC or analytical grades were purchased from Fisher Scientific (Pittsburgh, PA,
USA). Purified pig liver FMO, which gave an electrophoreticallysingle protein band, was kindly
supplied byDr D. M. Ziegler of the Universityof Texas, Austin.
Incubation procedures
Purified pig liver FMO was reconstituted in phosphate buffer (2 mm, pH 7.4) at a protein
ꢁ
1
concentration of 4 mg ml . Incubations were carried out in duplicate with the reconstituted enzyme
in 25 ml Erlenmeyer flasks at 378C using a shaking water bath in three separate occasions (n ¼ 3). A
typical incubation mixture consisted of a cofactor solution (0.5 ml), reconstituted enzyme (1 ml,
ꢁ
.1 mg ml protein) and substrate (20 ml) in acetone plus 0.480 ml distilled water (0–5 mm) in a total
+
1
0
volume of 2 ml. The cofactor solution consisted of NADP (1 mm), glucose 6-phosphate (5 mm),
glucose 6-phosphate dehydrogenase (1 unit) and magnesium chloride (10 mm, 50% w/w aqueous
solution) in sodium phosphate buffer (0.2 m, pH 7.4). Metabolism was initiated bythe addition of the
reconstituted enzyme and terminated by adding NaOH (0.5 ml, 0.1 m) to the incubation mixture. The
purified enzyme was omitted in control incubations. In another series of incubation flasks, EMS, CPMS
and DPS were incubated with the purified enzyme and a modified cofactor solution containing
ꢁ
1
superoxide dismutase (1000 units/flask) and catalase (11 000 units mg
protein). The metabolic
inhibition reactions were carried out at 378C in a shaking water bath using the optimum incubation
time of 15 min for EMS, CPMS and DPS. A saturating substrate concentration of 2.5, 0.5 and 0.25 mm
for EMS, CPMS and DPS, respectively, was used routinely for the metabolic inhibition studies. The
biologicallyformed sulphoxides were extracted and measured bychromatographic assaymethods.
Sample preparation and construction of calibration curves
The levels of the sulphoxide of EMS in the incubation mixture were monitored bya gas
chromatographic assaymethod. To the incubation samples, sodium h dy roxide (50 ml, 0.1 m), the
ꢁ
1
internal standard, n-propyl sulphone (10 ml, 1 mg ml ) and sodium chloride (0.1 g) were added. The
samples were then extracted with dichloromethane (2 Â 5 ml). The pooled extracts were evaporated to
dryness, reconstituted in 20 ml methanol and aliqutos (5 ml) of the reconstituted extracts injected onto the

8
6
I. P. Nnane and L. A. Damani
GLC column. The calibration curves for quantitation of EMSO were constructed byspiking var yi ng
ꢁ
1
amounts (0–50 mg ml ) into extraction tubes containing inactivated enzyme preparation, EMS (2.5 mm,
ꢁ1
cofactors and the internal standard, n-propyl sulphone (10 ml, 1 mg ml ), in a total volume of 2 ml of
incubation mixture.
The calibration curves for quantitation of CPMSO were constructed byspiking varying amounts (0–
ꢁ
0 mg ml ) into extraction tubes containing inactivated enzyme preparation (1 ml), CPMS (0.5 mm),
1
1
5
ꢁ
cofactors and the internal standard, CPPSO
2
(20 ml, 1 mg ml ), in a total volume of 2 ml incubation
mixture. The analytes were extracted with diethyl ether (2 Â 5 ml) using a mechanical bench test tube
shaker for 10 min followed bycentrifugation at 400 g for 10 min. The organic layers were evaporated to
dryness in a water bath at 408C. The extracts were reconstituted in a small volume (50 ml) of methanol
and an aliquot (10 ml) of the concentrate was injected onto the HPLC column. The ratios of the peak
area of the sulphoxide divided bythe peak area of the internal standard were plotted against actual
concentrations of the sulphoxide in the mixture. Similarly, the calibration curves for DPSO were
ꢁ
1
constructed byspiking var yi ng amounts (0–50 mg ml ) into extraction tubes containing inactivated
enzyme preparation (1 ml), DPS (0.5 mm), cofactors and the internal standard, CPPSO (20 ml,
mg ml ), in a total volume of 2 ml incubate. The mixtures were extracted with diethyl ether
2
ꢁ1
1
(
2 Â 5 ml) using a mechanical bench test tube shaker for 10 min followed bycentrifugation at 400 g
for 10 min. After HPLC analysis, the peak areas for DPSO and the internal standard were measured for
the construction of calibration curves as described for CPMSO above.
GC method
Gas chromatographywas performed on an HP 5840A gas chromatograph (Hewlett-Packard Co.,
Avondale, PA, USA), equipped with a flame ionization detector and a glass column (200 Â 0.4 cm).
Separation of EMSO and n-propyl sulphone was achieved on a glass column packed with 10%
Carbowax 20 M on Chromosorb W (Hewlett-Packard). The optimum working conditions for analysis
ꢁ1
of EMSO and n-propyl sulphone were: nitrogen flow rate, 30 ml min , injection port temperature,
008C, column temperature, 1708C; and detector temperature, 2508C.
2
HPLC method
1
The high-performance liquid chromatographic (HPLC) system used consisted of an hp HPLC
system (series 1050) equipped with a multichannel solvent delivery system coupled to an autosampler
and a photodiode arraydetection s ys tem operated at 260 nm (Hewlett Packard). Chromatographic
separation and quantitation of CPMS, DPS and their corresponding sulphoxides was achieved using a
1
reversed-phase Spherisorb 5ODS column (15 Â 0.46 cm) obtained from HPLC technology(Maccles-
field, UK). The analytical column was protected by a guard column packed with pellicular ODS
packing material. The mobile phase was composed of water and tetrahydrofuran (THF) (45:55 v/v) at a
ꢁ1
flow rate of 0.5 ml min for the studyof CPMS sulphoxidation. For DPS sulphoxidation studies, the
ꢁ
1
mobile phase composition was changed to THF/water (50:50 v/v) at a flow rate of 0.5 ml min
.
Data analysis
1
1
Least-squares regression analysis on SigmaPlot for Windows (SPSS Science Chicago, IL, USA)
was used to evaluate calibration curves and the enzyme kinetics. The Lineweaver–Burk transformation
of the Michaelis–Menten rate equation was used to evaluate the enzyme kinetics in this study:
ꢀ
ꢁ 1
1
Km
1
¼
þ
;
V
Vmax ½SŠ Vmax
where [S] is the concentration of the substrate, V is the rate of sulphoxide formation, Vmax is the
maximum rate of sulphoxide formation and K is the substrate concentration when one-half of the
maximum rate of sulphoxide formation is attained. K , Vmax and K were calculated byleast-squares fit
m
m
i
1
to the kinetic equations. One-wayanalysis of variance (ANOVA) on SigmaStat for Windows was used
to obtain probabilities (p) and compare different treatment groups. The Bonferroni post-hoc test was
used for the determination of the level of significance. p < 0:05 was considered as statistically
significant.
Result and discussion
EMSO (R ¼ 3.9 min) and the internal standard, n-propyl sulphone
t
(
R ¼ 7.5 min) were separated satisfactorilybyusing the 10% Carbowax 20 M on
t
Chromosorb W column and the calibration curve for EMSO was linear and
reproducible over the range examined (r ꢂ 0:997). The inter- and intradayvari-

Sulphoxidation of simple sulphides
87
ation at the lower concentrations was <10%, and <5% at the higher concentra-
tions.
Satisfactoryseparation of CPMSO ( R ¼ 4.8 min) and the internal standard,
t
CPPSO (R ¼ 9.6 min), was achieved byusing the reversed-phase HPLC con-
2
t
ditions described. Similarly, satisfactory separation of DPSO (R ¼ 5.6 min) and
t
the internal standard, CPPSO (R ¼ 10.2 min), was achieved byusing the re-
2
t
versed-phase HPLC conditions described. The calibration curves for measuring
the sulphoxide of CPMS or DPS were linear (r ꢂ 0:995) in the range 0–50 mg mlꢁ
and the assaymethods were reproducible with low inter- and intra-assayvariation
of <13.5%. The assaymethods were simple, fairlyrapid and allowed accurate
estimation of enzymically formed sulphoxides of CPMS and DPS in vitro.
The purified pig liver FMO catalysed the sulphoxidation of the model
sulphides, EMS, CPMS and DPS to their corresponding sulphoxides under the
incubation conditions used. The sulphoxidation of EMS, CPMS and DPS
increased linearlywith respect to time (0–30 min) and protein concentration
1
(
0.02–0.2 mg ml 1) and followed a typical Michaelis–Menten profile with respect
ꢁ
to substrate concentration (0–5 mm). A Lineweaver–Burk plot of EMS sulphox-
idation was linear (figure 2). The apparent Km and Vmax for the sulphoxidation of
ꢁ
1
ꢁ1
EMS were 1.38 ꢀ 0.05 mm and 78.74 ꢀ 3.9 nmoles mg protein min , respect-
ively. Similarly, Lineweaver–Burk plots of CPMS and DPS sulphoxidation were
linear (figures 3 and 4). The estimated apparent Km and Vmax for the sulphoxida-
ꢁ
1
ꢁ1
tion of CPMS were 0.185 ꢀ 0.03 mm and 103 ꢀ 5.0 nmoles mg protein min ,
respectively. The estimated apparent Km and V for the sulphoxidation of DPS
max
ꢁ
1
ꢁ1
were 0.068 ꢀ 0.002 mm and 49.26 ꢀ 2.05 nmoles mg protein min , respectively.
Figure 2. Lineweaver–Burk plots of ethyl methyl sulphide (EMS) sulphoxidation and the inhibitory
effects of 1-naphthylthiourea (NTTU) on EMS sulphoxidation by purified pig liver flavin-
ꢁ
1
containing monooxygenase. The incubation conditions were protein content ¼ 0.1 mg ml
;
incubation time ¼ 15 min and EMS concentrations ¼ 0.1–5 mm. Inhibitor concentrations were
0
.1, 1 and 5 mm. Values are the means of three separate experiments (n ¼ 3). The spread was 5–
10% of the means.

8
8
I. P. Nnane and L. A. Damani
A significant (p < 0:05) and concentration-dependent reduction of the sulph-
oxidation of these simple sulphides was observed with the addition of 1-
naphthylthiourea, a known inhibitor of FMO, in the incubation medium (figures
2
–4). The results indicate that 1-naphthylthiourea competitively inhibited the
sulphoxidation of EMS bypurified pig liver FMO with a K ¼ 8.49 ꢀ 0.51 mm
i
(figure 2). Analysis of the sulphoxidation of CPMS and DPS by purified pig liver
FMO in the presence of 1-naphthylthiourea suggest that the inhibitor non-
competitivelyinhibited the sulphoxidation of CPMS and DPS with K of
i
1
0.95 ꢀ 0.78 and 6.8 ꢀ 0.34 mm, respectively(figures 3 and 4). On the other
hand, incorporation of catalase and superoxide dismutase into the incubation
media produced no appreciable inhibition (p > 0:05) of the observed sulphoxida-
tion of these simple sulphides (table 1). Non-enzymatic oxidation of the sulphides
was negligible since the incorporation of catalase and superoxide dismutase into
the incubation media produced no statisticallysignificant inhibition of the ob-
served sulphoxidation of the compounds. If reactive oxygen species played a
significant role in the oxidation of these substrates then the inclusion of catalase
and superoxide dismutase into the incubation medium would have markedly
inhibited the metabolic reactions.
Previous studies have reported that the sulphoxidation of EMS byrat liver
microsomes is mediated predominantlybythe flavin-containing monoox yg enase
(Nnane and Damani 1999). Our present findings with purified pig liver FMO
support the contention that FMO is responsible for the sulphoxidation of the more
nucleophilic aliphatic sulphide, such as EMS where the sulphur atom resides
adjacent to alkyl groups, to its corresponding sulphoxide. In a separate report, it
Figure 3. Lineweaver–Burk plots of 4-chlorophenyl methyl sulphide (CPMS) sulphoxidation and
the inhibitoryeffects of 1-naphthylthiourea (NTTU) on the sulphoxidation of CPMS bypurified
pig liver flavin-containing monooxygenase. The incubation conditions were protein con-
ꢁ
1
tent ¼ 0.1 mg ml ; incubation time ¼ 15 min and CPMS concentrations ¼ 0.05–2.5 mm. Inhibi-
tor concentrations were 0.1, 1 and 5 mm. Values are the means of three separate experiments
(n ¼ 3). The spread was 5–10% of the means.

Sulphoxidation of simple sulphides
89
Figure 4. Lineweaver–Burk plots of the sulphoxidation of diphenyl sulphide (DPS) and the inhibitory
effects of 1-naphthylthiourea (NTTU) on the sulphoxidation of DPS by purified pig liver flavin-
ꢁ
1
containing monooxygenase. The incubation conditions were protein content ¼ 0.1 mg ml
;
incubation time ¼ 15 min and DPS concentrations ¼ 0.05–0.25 mm. Inhibitor concentrations
were 0.1, 1 and 5 mm. Values are the means of three separate experiments (n ¼ 3). The spread
was 5–10% of the means
Table 1. Effects of catalase and superoxide dismutase on the
sulphoxidation of ethyl methyl sulphide (EMS), 4-chlorophenyl
methyl sulphide (CPMS) and diphenyl sulphide (DPS) by purified
pig liver flavin-containing monooxygenase.
ꢁ1
ꢁ1
Enzyme activity (nmoles mg protein min )
Purified FMO þ
Substrate
Purified FMO
catalase/superoxide dismutase
EMS
CPMS
DPS
44.5 ꢀ 4.08
73.88 ꢀ 5.20
41.77 ꢀ 2.10
40.95 ꢀ 3.28*
67.97 ꢀ 5.44*
40.12 ꢀ 2.40*
Incubation conditions were protein content ¼ 0.1 mg mlꢁ ; incubation
1
time ¼ 15 min. Substrate concentrations were 2.5 mm for EMS, 0.5 and
0
.25 mm for DPS metabolism studies.
*
p > 0:05. Data are the means (ꢀ SD) from three separate experiments
(
n ¼ 3).
was observed that the sulphoxidation of CPMS is mediated byboth c yt ochrome
P450 and FMO in rat liver microsomes (Nnane and Damani 2001). This finding
was in agreement with another studywhere it was reported that the sulphoxidation
of a simple alkylaryl sulphide was mediated by both cytochrome P450 and the
flavin-containing monooxygenase (Waxman et al. 1982). Thus, the findings in the
present studywith purified pig liver FMO demonstrate that FMO has a role in the
sulphoxidation of sulphides of intermediate nucleophilicitysuch as CPMS, where
the sulphur atom resides adjacent to both alkyl and aryl groups.

9
0
I. P. Nnane and L. A. Damani
The formation of the sulphoxide of DPS was also dependent on the presence of
the purified pig liver FMO. The effects of catalase and superoxide dismutase on
the S-oxidation of DPS bypig liver FMO was evaluated to rule out the
participation of reactive oxygen species in this metabolic reaction. The sulphox-
idation of DPS bypig liver FMO was not significantly( p > 0:05) affected bythe
addition of catalase or superoxide dismutase in the incubation medium (table 1).
These results indicate that reactive oxygen species were not involved in the
oxidation of DPS. On the other hand, addition of with 1-naphthylthiourea, an
inhibitor of FMO, in the incubation mixture caused a significant decrease
(
4
p < 0:05) in DPS sulphoxidation in a concentration-dependent manner (figure
). These results suggest that DPS is catalysed to its corresponding sulphoxide by
purified pig liver FMO. Previous studies have indicated that the S-oxidation of
simple diaryl sulphides was mediated mainly by cytochrome P450 monooxygenase
(Hoodi and Damani 1984, Nnane and Damani 2002). In contrast, the present
studyclearlydemonstrates a significant role for the FMO in the sulphoxidation of
a diaryl sulphide such as DPS. Therefore, it would appear that the FMO
contributes to the sulphoxidation of less nucleophilic thioethers such as DPS,
where the sulphur atom resides adjacent to aromatic groups, in addition to its
established role in the S-oxidation of nucleophilic compounds bearing the sulphur
atom.
In conclusion, our studies demonstrate the capacityof purified pig liver FMO
to catalyse the oxidation of both nucleophilic substrates such as EMS and
substrates of intermediate nucleophilicitysuch as CPMS and also contributes to
the S-oxidation of electrophilic substrates such as DPS. These simple sulphides
maybe used as probes for assessing the relative contribution of the microsomal
monooxygenases to sulphoxidation reactions and the information generated on the
sulphoxidation of these simple sulphides bythe pig liver FMO maybe used to
predict the S-oxidation of more complex xenobiotics bearing the thioether func-
tional groups in humans.
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