Electrochemical versus Enzymatic in Vitro Oxidations of 6-propyl-2-thiouracil: Identification, Detection, and Characterization of Metabolites

Article abstract of DOI:10.1021/acs.jpcb.7b07404

6-Propylthiouracil, PTU, is a well-known antithyroid drug that has been the mainstay of treatment of Graves' disease. It is, however, also associated with liver toxicity and idiosyncratic toxicity. These toxicities are generally associated with metabolites derived from its bioactivation. In this manuscript, bioactivation of PTU was studied via two separate techniques: Electrochemical oxidation and through the use of human liver microsomes. The aim of this work was to compare the bioactivation products of these two techniques. The electrochemical technique was studied online with a mass spectrometer, EC/ESI/MS. The microsomal oxidations were studied in tandem with liquid chromatography. The EC/ESI/MS technique was devoid of the normal reducing biological matrix prevalent in microsomal incubations. The predominant product at 400 mV was the dimeric PTU species with negligible formation of other metabolites. At higher potentials, complete desulfurization of PTU was observed with formation of sulfate. No sulfonic acid was observed, suggesting that the cleavage of the C-S bond was effected at the sulfinic acid stage, releasing a highly reducing sulfur species which is known to give rise to genotoxicity. The microsomal oxidations, surprisingly, showed formation of the unstable sulfenic acid, the S-oxide. Further incubation showed both the sulfinic and sulfonic acids. None of the systems showed any adducts with nucleophiles such as glutathione, showing that none of the reactive metabolites were stable enough to be adducted to nucleophiles in both the biological matrix and the electrochemical oxidizing environment.

Full text of DOI:10.1021/acs.jpcb.7b07404

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Article
Electrochemical versus Enzymatic in Vitro Oxidations of 6-propyl-2-
thiouracil: Identification, Detection and Characterization of Metabolites
Kudzanai Chipiso, and Reuben Hazvienzani Simoyi
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07404 • Publication Date (Web): 01 Nov 2017
Downloaded from http://pubs.acs.org on November 2, 2017
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Electrochemical versus Enzymatic in Vitro Oxidations of 6-propyl-2-thiouracil:
Identification, Detection and Characterization of Metabolites
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by
Kudzanai Chipisoᶧ and Reuben H. Simoyi*ᶧǂ
ᶧDepartment of Chemistry, Portland State University,
Portland, OR 97207-0751, U.S.A.
ǂSchool of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus,
Durban 4014. South Africa
Corresponding author email address: rsimoyi@pdx.edu . Phone: 503-725-3895
The authors declare no competing financial interest.
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ABSTRACT: 6-Propylthiouracil, PTU, is a well-known antithyroid drug that has been the
mainstay of treatment of Graves disease. It is, however, also associated with liver
toxicity and idiosyncratic toxicity. These toxicities are generally associated with
metabolites derived from its bioactivation. In this manuscript, bioactivation of PTU was
studied via two separate techniques: electrochemical oxidation and through the use of
human liver microsomes. The aim of this work was to compare the bioactivation
products of these two techniques. The electrochemical technique was studied on-line
with a mass spectrometer, EC/ESI/MS. The microsomal oxidations were studied in
tandem with liquid chromatography. The EC/ESI/MS technique was devoid of the
normal reducing biological matrix prevalent in microsomal incubations. The
predominant product at 400 mV was the dimeric PTU species with negligible formation
of other metabolites. At higher potentials, complete desulfurization of PTU was
observed with formation of sulfate. No sulfonic acid was observed, suggesting that the
cleavage of the C – S bond was effected at the sulfinic acid stage, releasing a highly
reducing sulfur species which is known to give rise to genotoxicity. The microsomal
oxidations, surprisingly, showed formation of the unstable sulfenic acid, the S-oxide.
Further incubation showed both the sulfinic and sulfonic acids. None of the systems
showed any adducts with nucleophiles such as glutathione, showing that none of the
reactive metabolites were stable enough to be adducted to nucleophiles in both the
biological matrix and the electrochemical oxidizing environment.
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INTRODUCTION
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6-propyl-2- thiouracil (PTU) is an antithyroid drug that was introduced close to seventy
years ago.1 This drug is effective in the treatment and management of Grave’s disease,
an autoimmune disease mediated by thyroid-stimulating immunoglobulins; resulting in
hyperthyroidism, which is over production of thyroid hormones.2-8 It works by inhibiting
the activity of thyroid peroxidase and blocks the conversion of thyroxine (T4) to
triidothyronine (T3).9-11 The action of PTU may partly depend on its ability to function as
a thiol as shown by tautomer forms below.
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Figure 1: Tautomers of PTU
Although effective, PTU has been associated with rare but severe idiosyncratic
toxicity, characterized by skin reactions, agranulocytosis, aplastic anemia, hepatitis and
cholestasis. 1, 12-16 PTU is identified as being the third most frequent cause of drug-
induced liver transplant in the United States.17 In 2008, a panel of experts extensively
reviewed adverse events reports and case reports of PTU-induced liver failure and
deaths; they recommended that PTU should not be used as first line of therapy in
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Grave’s disease.18 In 2009 the Food and Drug Administration (FDA) issued a black box
warning about severe liver injury associated with treatment using PTU.19, 20
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Hepatotoxicity of therapeutic drugs is not a new phenomenon in drug therapy. A
number of drugs have already been withdrawn from the market after cases of severe
toxicity were noted.21, 22 Bioactivation of drugs resulting in reactive metabolites is
considered as the initial step towards drug-induced organ damage23. Presumably, the
hepatotoxicity associated with PTU is due to its reactive metabolites in the liver, these
could be radicals or electrophiles. However, as of now, there are no reports on PTU
reactive metabolite(s) formation in the liver and the role of formed intermediates in the
hepatotoxicity induced by this drug is ambiguous24. There has been a variety of
metabolites observed from PTU metabolism. Thiols are metabolized to sulfenic acids,
which are unstable and readily undergoes oxidation to their stable oxides; sulfinic and
sulfonic acids or combine with nucleophiles. Whereas there have been some studies on
electrochemical oxidation of PTU, a lot of these studies were focusing on modifying
electrodes to enhance electrocatalytic oxidation of PTU using cyclic voltammetry and
differential pulse voltammetry.25, 26 Electrocheimcal oxidation of PTU is complicated by
large overpotentials. Modified electrodes have been used due to their catalytic
properties that lower the anodic overpotential and enhance the rate of electron transfer
for the electrochemical oxidation of PTU. In the present work, the oxidation of PTU is
investigated using an electrochemical flow through cell that uses porous graphite
working electrode and combines electrochemistry and mass spectrometry. Metabolites
generated by electrochemical oxidation were compared to those that were identified
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from standard microsomal oxidation. Slight differences were noted in the identity of the
metabolites.
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MATERIALS AND METHODS
Chemicals. Reagent grade 6-propyl-2-thiouracil, PTU, reduced glutathione, N-
acetylcysteine, acetic acid and ammonium acetate were obtained from Sigma Aldrich
and were used without further purification. Ammonia, methanol, acetonitrile,
phosphate monobasic and phosphate dibasic were from Fischer Chemicals. Water for all
experiments was purified using a Barnstead Sybron Corp. water purification unit capable
of producing both distilled and deionized water (Nanopure). Solvents used for
electrochemical oxidation and mass spectrometry were HPLC grade.
On-line EC/ESI-MS Electrochemical Oxidation of Analytes. Experiments were
carried out in alkaline medium. The analyte was initially dissolved in alkaline medium
where it was more soluble. The media utilized a 20% methanol with 80 % 20 mM
ammonium buffer solution (pH 10.2). Electrochemical oxidations were performed using
Thermo Scientific Dionex 5150 Synthesis CellTM equipped with a flowꢀthrough graphite
working electrode, solid state palladium reference electrode and a Palladium counter
electrode (see Figure 2). The cell potential was controlled using Thermo Scientific
Dionex Coulochem III electrochemical detector. The cell outlet was interfaced into a
mass spectrometer inlet for onꢀline analysis using PEEK tubing. To prevent electrical
damage to the detector and cell; as well as shock, the synthesis cell and the detector were
decoupled from the high voltage of the mass spectrometer using a high voltage
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decoupling union kit as described before.27 Samples were infused through a syringe
pump at a flow rate of 10 µL/min for onꢀline experiments. Mass spectra of the
electrochemical oxidation metabolites, were acquired on a highꢀresolution (m/ꢀm = 30
000) Thermo Scientific LTQꢀOrbitrap Discovery mass spectrometer (San Jose, CA)
equipped with an electrospray ionization source. The MS ESI source parameters were set
as follows: spray voltage (kV), 2.5 in negative mode and 4.5 in positive mode; spray
current (ꢁA), 1.96; sheath gas flow rate, 20; auxiliary gas flow rate, 0.01; capillary
voltage (V), −16; capillary temperature (°C), 300; and tube lens (V), −115. Detection was
carried out in both the negative ionization mode and positive (−ESI) for 4 min. The
detection parameters were set up as follows: Analyzer; FTMS, positive and negative
polarity; mass range; normal, resolution; 30 000, scan type; centroid.
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Microsomal Enzymatic Oxidation. Human liver microsomes were obtained from
Corning. A well-established protocol for microsomal incubation (provided from Corning
upon purchase of the microsomes) was followed with minor changes to suit the
experiment and substrates involved. The microsomal fractions were stored at -80 °C and
thawed immediately before the experiment. The following quantities were used.
Initially, 713 µL of purified water, 200 µL of 0.5 M potassium phosphate buffer pH 7.4,
50 µL NADPH regenerating system solution A (solution A contains NADP+ and Glucose-6-
phosphate), 10 µL NADPH regenerating system solution B ( Solution B contains Glucose-
6-phosphate dehydrogenase) 2 µL substrate in solvent, making a final substrate
concentration of 10µM, were combined before adding microsomes. When combined,
solutions A and B provide the regenerating system for all NADPH-requiring oxidase
assays. After warming in a 37 °C water bath for five minutes, the reaction was then
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initiated by adding 25 µL (0.5 mg ) liver microsomes and then was mixed and returned
to a 37 °C water bath. After 60 minutes a 100 µL aliquot from the incubation was
withdrawn and the reaction was terminated by adding 100 µL acetonitrile. This was
then mixed and placed on wet ice followed by centrifuging at 10,000 Xg for three
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minutes. The supernatant from the protein pellets was withdrawn for analysis using
LC/MS. Incubations without NADPH and microsomes were used as controls, to ensure
that formation of metabolites was dependent on HLMs and NADPH.
LC-MS Analysis. The metabolites produced by incubation of PTU were collected
and analyzed by LCꢀMS. The separation of the metabolites was achieved using a reverse
phase Zorbax Eclipse Plus 3.5 µm, C8 column. Colum dimensions were as follows: 100
mm x 2.1 I.D. The flow rate was set at 200 µL/min and the injection volume was 20
µL/min. The gradient separation was performed by two solvents. Mobile phase A: 0.1%
acetic acid in water and mobile phase B: 0.1% acetic acid in methanol. The applied
gradient profile was 10% B for 5 minutes, followed by 30 % for another 5 minutes, and
then 50:50 for two minutes and finally 90% B for the last six minutes. The column was
operated at 35 °C. The MS based detection was carried out it positive ion mode using
both FTMS and ITMS detection.
RESULTS AND DISCUSSION
Electrochemical Oxidation of PTU. Electrochemical oxidation of PTU was carried out
using the EC/ESI/MS arrangement shown in Figure 2.
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Figure 2. Schematic of EC/ESI –MS system. Coulochem III Electrochemical Detector controls
potential of the flow through cell. Samples were infused at 10 µL/min.
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169.04500
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174.95677
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m/z
Figure 3 ESI (-) MS, with cell turned off 0 mv obtained for 0.1 mM PTU in 20 mM
ammonium phosphate buffer, 20% MeOH, pH 10, Flow rate 10 µL/min.
Control experiments were initiated with the cell turned off at zero potential.
Figure 3 clearly shows a clean ESI-spectrum of PTU, that was obtained in the negative
mode. The only peak that is evident from the spectra above is the substrate PTU with
m/z 169.04
The presence of a single peak from the solvent electrolyte mixtures shows that
there were no other reactions or contaminants prior to the electrochemical oxidation.
Contaminants as well as other reactions in the electrolyte mixture are usually confused
with modifications that might take place on the compound of interest. As such, any
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products that emerged when the reaction was initiated by applying potential could thus
be attributed to transformations due to oxidation of PTU in the electrochemical cell.
When the cell was turned on, the voltage was gradually increased with a ramp of 100
mV at each step, each time. Emergence of additional peaks were evident at 400 mV.
This potential was enough to initiate oxidation of PTU to a disulfide and sulfinic acid, as
shown in Figure 4. PTU disulfide was the dominant peak in both acidic and alkaline
medium. However, in acidic medium there were other spurious peaks that could not be
easily assigned. This demonstrates the difficulty in choosing suitable solvents for EC-MS
system, to avoid ion suppression.
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337.08059
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169.04502
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Figure 4. ESI-MS spectra generated with the cell potential at 400 mV.
Figure 4 shows a strong peak of PTU-disulfide, which is the major product of
oxidation at the given potential of 400 mV. Dimeric species from thiols are usually
unreactive compared to parent thiols, hence they accumulate. Surprisingly, the
electrochemical oxidation of PTU was achieved at very low potentials when compared
to what has been reported previously in literature.28 Sartori et al, reported higher
electrode potentials of 1.42 V for oxidation of PTU to its dimer using cyclic
voltammetry.28 Electrode potential, (Em), is pH-dependent, and has been shown to
decrease at approximately 0.06 V/pH for a simple thiol such as glutathione. Therefore,
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as the pH is increased, the potential required to effect oxidation decreases.29 The data
below suggests the possibility of two competing reaction pathways. Thiol oxidation can
proceed through two competing pathways. Abstraction of a single electron is relatively
easy leading to thiyl radicals. The two electron pathway result in formation of a sulfenic
acid. These two intermediates participate in further reactions resulting in metastable
products.30 Thus the thiyl radical might initiate chain reactions resulting in disulfides.
This is a coupled proton-electron transfer process resulting in a radical that dimerizes to
a disulfide as shown in Scheme 1.
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Scheme 1 Proposed oxidation scheme for PTU and its dimerization to a disulfide
compound.
There is a relatively weak peak at m/z 201 which is attributable to a sulfinic acid.
In theory one would expect to observe the oxides of PTU in the mass spectrum. Thiols
are known to undergo oxidation through the sulfenic acid which further oxidizes to
more stable sulfinic and sulfonic acids, respectively31, 32
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RSH
RSOH
sulfenic acid
RSO2H
Thiol
sulfinic acid
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However, there is no evidence of formation of sulfenic acid, or the S-oxide. The
sulfenic acid may disproportionate to its meta stable S-oxide. Sulfenic acids exhibit
potent electrophilic and relatively weak nucleophilic reactivity.33 Thus, in small
molecules such as PTU, this dual behavior can lead to self-condensation in which one
sulfur atom functions as a nucleophile and the second as an electrophile to yield a
thiolsulfinate ester:
The self-condensation reaction is facilitated by intermolecular hydrogen
bonding. At a pH > 8, which is the case in this experiment, the thiolsulfinate esters
disproportionate to form salts of sulfinic acid and disulfide.34 This reaction can thus
explain both the presence of sulfinate and the dimer observed in Figure 4. Trapping
experiments were also conducted in PTU oxidations. Unlike in oxidation of
methimazole,35 there were no conjugates that formed between PTU and nucleophiles.
Presumably, the reaction proceeds through formation of the dimer, the dimer then
would further be oxidized to sulfinic acid. Regardless of which pathway dominates, both
mechanisms have potential to result in reactive intermediates
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Effect of Oxidation Potential. An analysis of ESI spectrum collected at higher
oxidative potentials of 800 mV shows two additional peaks that were observed on the
mass spectrum. Figure 5 shows that sulfur was fully oxidized, from -2 to +6, to yield
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2-
sulfate (SO4 ) shown, with m/z 96. Effluent from the cell was reacted with barium
chloride, resulting in formation of BaSO4. There was no evidence of formation of PTU-
sulfonic acid from the ESI mass spectra. Hydrolysis of sulfinic acid, should result in the
cleavage of the C-S bond to give a urea-type residue, R1R2C=O and an unstable sulfur
species HSO2 . HSO2 is readily oxidized to bisulfite (HSO3 ).36
-
-
-
R1R2CSO2H + H2O ꢀ R1R2C=O + HSO2 + H+
R2
R3
-
HSO2 + H2O ꢀ HSO3 + 2H+ + 2e-
-
-
-
HSO3 is, in turn, easily oxidized to sulfate. One would expect a significant
amount of the urea-type organic residue if hydrolysis was the major pathway. However,
there are only traces (of propyl uracil) at m/z 153 and an unidentified metabolite with
m/z 135. If this organic residue is not stable, it would further hydrolyze through ring
opening giving smaller fragments. A decrease of 32 amu is apparent from the initial
mass of substrate. The exact mechanism from the sulfinic acid to sulfate and an organic
residue can only be speculative, but involves no further oxidation past the formation of
sulfate. Non solvent assisted cleavage of C-S bond which is either heterolytic or
homolytic cleavage should result in loss of sulfur as sulfite and sulfite radical anion
respectively, from sulfinic acid37. The organic residue observed is shown with the peak
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at m/z of 137 thus this is 6-propyl-2, 3-dihydropyrimidin-4(1H)-one. The spectrum below
still shows the dimer at m/z 168 which is the symmetrical PTU dimer.
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Figure 5. ESI-MS spectra generated with the cell potential at 800 mV.
The one electron pathway, resulting in thiyl radicals, appears to be the most
dominant mechanism here. Radicals play a very important role in induced oxidative
stress and subsequent liver injury. Waldhauser and co-workers investigated the
oxidation of PTU in activated neutrophils.38 PTU was observed to be oxidized by
myeloperoxidase39 giving sulfite as one of the products, as indicated above, which can
subsequently be oxidized to sulfate. If oxidation of PTU, proceeds through
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desulfurization, forming sulfite radical, one would expect the sulfite to undergo further
reactions which may or may not lead to reactive metabolites. Apparently it has been
shown that sulfite is oxidized by myeloperoxidase to free radicals. These prooxidant
radicals, such as the reactive sulfur trioxide (SO3•−), peroxymonosulfate (−O3SOO•)40 and
sulfate (SO4•−) anion radicals, can damage proteins and oxidize them to protein radicals,
with consequences of allergic reactions41. Whereas some studies reported that there
have been no reactive metabolites formation in the liver, however, in our studies in this
manuscript, some intermediates e.g., 6-propyl-2,3-dihydropyrimidin-4(1H)-one ,
(propyluracil) and other unidentified metabolites, with prooxidant radicals were
echoed in the electrochemical system. Some of the intermediates have been proposed
but have not been observed in previous in vivo and in vitro studies. Radicals can also
generate toxic effects through oxidative stress without forming covalent adducts with
biomolecules.42 This forms the basic framework for the danger hypothesis that leads to
idiosyncratic toxicities.
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In Vitro Enzymatic Oxidation of PTU. The metabolism of PTU was studied on the
basis of accepted standard in vitro techniques involving incubation of target compound
with liver microsomes. The experiments reported here were performed using human
liver microsomes. The results obtained from the liquid chromatography separation with
mass spectrometry detection are shown below.
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Oxidation of PTU by microsomes was complex. Identification of the products
from metabolism was complicated by the presence of the biological matrix and a range
of possible metabolites. Although not quite abundant, Figure 7 shows spurious peaks on
the ESI-spectrum, some of which were unidentified. However, this observation does not
really point to an inferior technique. Instead, it does show that a variety of metabolites
are possible from biological oxidations versus EC-MS
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Figure 6. LC-MS chromatograms form metabolites generated by incubation with human
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Figure 6 shows the chromatogram, extracted from ion filtering using Xcalibur
software. Retention time of 14.30 shows the unconverted substrate, PTU with m/z
171.13 shown in Figure 7. Another peak on the chromatogram, with retention time of
5.81 was associated with the unconverted substrate; this could be due to tautomer of
PTU, resulting in a more polar ion form of PTU. This analysis is further complicated by
appearance of another small peak that co-eluted at this same retention time. The peak
had a jump of 16 amu from the substrate, thus [M + O + H] with at m/z 186.22, the
putative PTU S-Oxide. The relatively low abundance of the peak, appears to show a
transient molecule that is derived from unstable sulfenic, moreover it is fleeting towards
sulfinic and sulfonic acids. In this biological matrix, the reactive sulfenic acid can react
with protein components of microsomes. Usually, after centrifugation, the protein pellet
will sediment and is not analyzed in LC-MS analysis, hence a diminished peak of the
remaining metastable S-oxide can be observed. Its polarity also fits very well to the
assigned retention time.
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Figure 7 ESI (+) mass spectrum showing product of oxidation from microsomal
incubation.
Although hepatotoxicity due to PTU has been known, it is surprising that there
have not been many studies on oxidation of PTU using microsomes. Most of the studies
conducted used peroxidases as oxidants, and as such there have not been known
reactive metabolites produced from microsomes. Studies on oxidation of PTU with
activated neutrophils showed that PTU-disulfide, PTU-sulfite/sulfinic acid and PTU-
sulfate/sulfonic acid were major products of oxidation.39. The toxicity of thionamides,
such as ethionamide has been associated with their bioactivation to reactive S-Oxides.43,
44 Therefore, hepatotoxicity due to PTU may also arise from the observed S-oxide, this
electrophilic metabolite it has the capacity to bind to liver cellular macromolecules
resulting in drug in damage to cells.
Mechanism of Oxidation. Metabolism by microsomes involves primarily the
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flavin-containing monooxygenases (FMO’s) and the Cytochromes P450 group of
enzymes. FMO’s are a family of drug-metabolizing enzymes that use FAD, NADPH and
molecular oxygen to catalyze the oxygenation of a large number of xenobiotics
containing ‘soft’ nucleophiles such as sulfur, phosphorus and nitrogen.45, 46
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Scheme 2 Proposed reaction scheme for PTU metabolism in microsomes.
The scheme above shows a simple mechanism for oxidation of PTU in
microsomal incubations. FMOs convert xenobiotics into polar metabolites by adding
oxygen so that they can subsequently be easily eluted through the kidneys.47 While
initially thought to be a single enzyme; FMO’s are now known to contain at least 11
isoforms, with 5 of them having been characterized to date.48, 49 It has been reported
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that FMOs oxidize thionamides and thiourea functional groups to intermediate
metabolites of sulfenic (RSOH) and sulfinic (RSO2H) acids which are responsible for
deactivating CYP450 enzymes.50, 51 As mentioned earlier on (vide supra), sulfenic acid is
a very unstable intermediate, giving rise to the metastable S-oxide. In presence of
excess oxidative power, the S-Oxide and sulfinic acid will further be oxidized to sulfonic
acid, as shown in figure 8.
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The standard procedure for trapping reactive metabolites was also carried out
using glutathione and n-acetyl cysteine as trapping agents. There were no detectable
adducts/conjugates in metabolic oxidation reactions of PTU. Sulfenic acid precedes
formation of S-oxide and is very reactive and is able to react with any nucleophile,
especially thiols. However, even if the intermediates were formed, their conjugation
with nucleophiles is not always facile, sometimes it requires enzymes, and such as
glutathione transferase to assist in conjugation.52
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Figure 8 ESI (+) MS spectra showing sulfinic acid and sulfonic acid as products of
oxidation of PTU.
PTU is less polar as compared to its products of oxidation, which co-eluted within
1.95 and 2.06 minutes. Figure 8 shows propylthiouracil sulfinic acid (PTU-SO2H) m/z
203.04 and propylthiouracil sulfonic acid (PTU-SO3H) m/z 218.94 and its sodium adduct
m/z 240.09 as the major products of microsomal oxidation. These are stable oxides for
oxidation of PTU. PTU-sulfonic acid/sulfonate, at m/z 218.94, [M +3O +H+] and 240.09,
[M +3O +Na+] exist in zwitterionic forms before being adducted to proton or sodium
respectively. Microsomal oxidations were carried at a pH of 7.4, close to neutral pH, and
thus this environment can afford such neutral structures. Molecules with such
structures have been observed from X-ray data. Chigwada et al,53 showed the existence
of this putative sulfonic acid zwitterion for tetra methyl thiourea sulfonic acid (TTTU-
-
SO3 ) (see Figure 9). Figure 9 shows the putative molecular ion as observed in ESI
spectrum at m/z 240.02.
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Figure 9. Putative zwitterion structures. On the left is the X-ray structure determined
by Chigwada et al in their seminal manuscript, on the right is the zwitterion expected for
PTU.
Figure 8 shows an undefined and unidentified metabolite at m/z 196.96. This
highlights some of problems that arise from microsomal oxidations. The matrix contains
different endogenous material which can co-elute with compounds of interests.
Contrary to this, it also shows diversity of metabolites that can be obtained from
microsomal oxidations.
Comparison of Electrochemical and Biological Metabolism. There were
variations in the nature and type of metabolites that have been observed from
electrochemical oxidation of the PTU compared to those observed and reported in
biological studies .54 Both Figures 7 and 8 show some unidentified peaks e.g., peak with
m/z 196 could not be assigned to any metabolite in the mixture. The electrochemical
process is a ‘cleaner’ oxidation environment than that afforded by microsomes. Thus the
electrochemical oxidation platform involves the most easily oxidizable part of the
molecule without any regard to other parameters present in bimolecular activations
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such as stereochemistry. Oxidation of thiols through formation of their disulfides is
relatively easy, compared to two electron process for the formation of an S-oxide.
Contrary to EC-MS system, microsomal oxidations show non-competing reaction
pathways. Successive formation of sulfur oxo-acids is through the well-established two-
electron transfer mechanism of FMO S-oxygenation. Microsomes contain CYPs as well,
which participate in functionalization reactions, and as such one would expect a variety
of metabolites. However, there are some other factors that might prevent this; e.g.,
thionamides are known to be enzyme inhibitors,55 and this has a large bearing on nature
of products that can be obtained from their biological oxidation using microsomes. The
kinetics for the EC-MS, two electron mechanism are quite sluggish. Electron
withdrawing groups such as oxygen in the ring for PTU may reduce the nucleophilicity of
S atom on the thiolate ion, making it difficult to abstract a second electron. A
comparison of EC-MS oxidations to microsomal oxidations shows there is a physiological
gap between the isolated microsomes and electrochemical cells. However, there were
similarities in type of metabolites obtained from EC-MS with those obtained using
peroxidase enzymes. Differences in metabolites may be attributed to experimental
conditions.
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CONCLUSION
Bioactivations, mechanism, products and intermediates of sulfur-based drugs have
always been difficult to determine. PTU oxidation is complex, characterized by both free
radical and two electron process mechanisms in electrochemistry and microsomal
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oxidations respectively. PTU oxidation resulted in a range of metabolites. The absence
of conjugates in either system, does not preclude formation of reactive metabolites.
Reactive metabolites, by their nature, are short-lived, and hence elusive. Their lifetimes
are very small. However, results show that an S-Oxide, which is electrophilic and
hepatotoxic, is produced in liver microsomal incubations. Toxicity of PTU may be
attributed to this reactive metabolite. Moreover, the mechanism entails two pathways
which may be involved in toxicity attributed to PTU. EC-MS technique complements the
microsomes, and is informative in evaluation of toxicities from drugs, as it gives an
alternative explanation to observed physiological effects.
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Acknowledgments:
This work was supported by a research grant from the National Science Foundation,
Grant number CHE 1056366 and a partial research professorship vote from the
University of KwaZulu-Natal.
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