THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 39, Issue of September 25, pp.

25079 –25088, 1998

© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Free Radical Intermediates of Phenytoin and Related Teratogens

PROSTAGLANDIN H SYNTHASE-CATALYZED BIOACTIVATION, ELECTRON PARAMAGNETIC RESONANCE
SPECTROMETRY, AND PHOTOCHEMICAL PRODUCT ANALYSIS*

(Received for publication, June 8, 1998)

Toufan Parman‡, Guoman Chen§, and Peter G. Wells‡¶i

From the ‡Faculty of Pharmacy and ¶Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 2S2,
Canada and the §Department of Clinical Studies, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Phenytoin and related xenobiotics can be bioacti- arene oxide (Fig. 1) (7); 2) the teratogenicity of structurally
vated by embryonic prostaglandin H synthase (PHS) to a similar anticonvulsants, such as trimethadione (Tridione) and
teratogenic free radical intermediate. The mechanism of its N-demethylated pharmacologically active metabolite,
free radical formation was evaluated using photolytic dimethadione, that lack the phenyl substituent necessary for
oxidation with sodium persulfate and by EPR spectrom- the formation of an arene oxide; and 3) the relatively low
etry. Characterization of the products by mass spec- embryonic activity of most cytochrome P450s during organo-
trometry suggested that phenytoin photolyzes to a ni- genesis (4, 5, 9), including CYP2C9, which is known to bioac-
trogen-centered radical that rapidly undergoes ring tivate phenytoin (10).
opening to form a carbon-centered radical. PHS-1 was

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We have investigated an alternative hypothesis involving
incubated with teratogen (phenytoin, mephenytoin, tri- the bioactivation of proteratogens by peroxidases such as pros-
methadione, phenobarbital, and major metabolites) or
taglandin H synthase (PHS)1 to teratogenic free radical inter-
its vehicle and the free radical spin trap a-phenyl-N-t-
mediates (Fig. 2) (3– 6, 9). PHS and related potential bioacti-
butylnitrone, and incubations were analyzed by EPR
vating enzymes such as lipoxygenases are present with high
spectrometry. There was no a-phenyl-N-t-butylnitrone
radical adduct in control incubations. For phenytoin, a activity in the embryo during organogenesis, the period of
putative unstable nitrogen-centered radical adduct and major teratologic susceptibility. Xenobiotic free radicals can
a stable carbon-centered radical adduct were detected. bind covalently to cellular macromolecules (DNA, protein) and
Free radical spin adducts also were detected for all can initiate the formation of reactive oxygen species (ROS) that
other teratogens and metabolites except carbamaz- cause oxidative stress and oxidative damage to DNA, protein,
epine. The PHS inhibitor eicosatetraynoic acid abol- and lipid. As detailed in the above reviews (3– 6, 9), there is
ished the free radical EPR signal. Incubation of 2*-deox- evidence in vivo, in embryo culture, and in vitro for embryonic
yguanosine with phenytoin and PHS-1 resulted in a PHS-catalyzed bioactivation of phenytoin to a free radical in-
5-fold increase in its oxidation to 8-hydroxy-2*-deox- termediate that initiates embryotoxic ROS formation. Phenyt-
yguanosine. This is the first direct chemical evidence for oin initiates hydroxyl radical formation and the oxidation of
PHS-catalyzed bioactivation of phenytoin and related embryonic DNA, protein, thiols, and lipid. Conversely, phenyt-
teratogens to a free radical intermediate that initiates oin-initiated oxidation of embryonic cellular macromolecules
DNA oxidation, which may constitute a common molec- and teratogenicity or embryotoxicity are reduced by PHS in-
ular mechanism of teratologic initiation. hibitors, free radical spin trapping agents, iron chelators, an-
tioxidants, and antioxidative enzymes, including GSH reduc-
tase, GSH peroxidase, superoxide dismutase, and catalase
Phenytoin (diphenylhydantoin; Dilantin) is a widely used (3– 6, 9, 11–13).
anticonvulsant drug that is teratogenic in animals and humans On the other hand, little is known about the chemical nature
(1–3). Several teratologic mechanisms have been proposed, in- of the putative free radical intermediate of phenytoin and re-
cluding the bioactivation of phenytoin by embryonic cyto- lated xenobiotics in biological systems. Phenytoin and other
chrome P450 to an electrophilic arene oxide reactive interme- hydantoins, as well as the structurally related succinimides,
diate that covalently binds to embryonic protein, thereby contain an imidyl group as shown in boxes in Fig. 1. The
altering cellular function (1– 6). However, these hypotheses are generation of imidyl radicals (a nitrogen-centered radical
not consistent with a number of published observations, includ- flanked by two acyl groups) from N-halohydantoins and N-
ing 1) the association of embryopathic activities of the struc- bromosuccinimide has been studied and used in synthetic
turally similar, asymmetric hydantoin anticonvulsants mephe- chemistry for a bromination process since 1942 (14). N-Bromo-
nytoin (Mesantoin) and its N-demethylated active metabolite succinimide undergoes photodecomposition to generate an imi-
nirvanol with the L-isomers that primarily do not form the dyl radical that opens to form a carbon-centered radical with an
isocyanate moiety (14 –20). The characteristic reactions for
both of these radicals are hydrogen abstraction and addition to
* This work was supported by a grant from the Medical Research double bonds (14, 16, 19, 20).
Council of Canada. A preliminary report of this research was presented
at the 35th annual meeting of the Society of Toxicology, Anaheim,
California, March 1996 (60). The costs of publication of this article were
1
defrayed in part by the payment of page charges. This article must The abbreviations used are: PHS, prostaglandin H synthase; AA,
therefore be hereby marked “advertisement” in accordance with 18 arachidonic acid; 29-dG, 29-deoxyguanosine; ETYA, 5,8,11,14-eicosa-
U.S.C. Section 1734 solely to indicate this fact. tetraynoic acid; HFSC, hyperfine splitting constant; HPLC, high per-
i To whom correspondence should be addressed: Faculty of Pharmacy, formance liquid chromatography; 8-OH-29-dG, 8-hydroxy-29-deox-
University of Toronto, 19 Russell St., Toronto, Ontario M5S 2S2, Can- yguanosine; PBN, a-phenyl-N-t-butylnitrone; ROS, reactive oxygen
ada. Tel.: 416-978-3221; Fax: 416-978-8511; E-mail: pg.wells@ species; HPPH, 5-(p-hydroxyphenyl)-5-diphenylhydantoin; MS, mass
utoronto.ca. spectrometry.

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25080 Free Radical Intermediates of Teratogens

FIG. 1. Structures of phenytoin and

related drugs and metabolites. Struc-
tural similarities are indicated by the
boxes. In vivo murine studies have shown
that the N-demethylated drugs and me-
tabolites are substantially more terato-
genic than their respective methylated
parent molecules, suggesting that in vivo
N-demethylation is a prerequisite for per-

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oxidase-catalyzed bioactivation to a terat-
ogenic reactive intermediate (3). P450, cy-
tochrome P450.

Given that chemical studies indicate that N-halohydantoins mine hydrochloride, sodium persulfate, benzophenone, and 29-deox-
can form a nitrogen-centered radical on their imidyl moiety and yguanosine were obtained from Sigma-Aldrich (Oakville, Canada); re-
distilled phenol was from Aldrich. Mephenytoin and nirvanol isomers
biochemical studies have shown that hydantoins and related
were gifts from Dr. A. Küpfer (Switzerland); trimethadione was a gift
compounds can initiate hydroxyl radical formation and oxida- from Abbott. 5,8,11,14-Eicosatetraynoic acid (ETYA) was a gift from
tion of macromolecular targets, we hypothesized that phenyt- Hoffmann-La Roche. All other reagents used were of analytical or
oin and its analogs can be bioactivated by PHS to an imidyl free HPLC grade.
radical that can undergo ring opening to generate a carbon-
centered free radical with an isocyanate group. These radicals Methods
may covalently bind to embryonic macromolecules with carbon- Photochemical Generation of Phenytoin Free Radical—A 3-ml solu-
carbon double bonds, such as DNA and protein, and/or initiate tion containing 100 mM phenytoin and 100 mM sodium persulfate in 6.0
embryonic ROS formation and oxidative macromolecular dam- mM NaOH (pH 11.8) was photolyzed at 300 nm for 0, 10, 20, 30, and 40
age, thereby initiating teratogenesis. This hypothesis was in- min in a Rayonet chamber. The reaction mixture of each time interval
vestigated using two approaches. The first involved the chem- was analyzed for product formation by HPLC equipped with a model
222 solvent delivery system (Scientific Systems, Inc.), a 5-mm Spheri-
ical characterization of products following the photolytic
sorb ODS II C-18 column (15 cm 3 4.6 mm, Jones Chromatography,
oxidation of phenytoin using sodium persulfate. The second Lakewood, CO), a model SPD-6AV UV/Vis detector (Shimadzu, Kyoto,
approach involved direct characterization of teratogen free rad- Japan), and an integrator (Chromapac model CR501; Shimadzu). The
ical intermediates by EPR spectrometry and phenytoin-initi- mobile phase consisted of 59% water, 1% glacial acetic acid, and 40%
ated DNA oxidation, following in vitro incubation with PHS. acetonitrile, at a flow rate of 1 ml/min. The product separation was
The results provide the first direct chemical evidence for PHS- performed at 240 nm.
Identification of Photolysis Products by Thin Layer Chromatogra-
catalyzed bioactivation of phenytoin and related proteratogens
phy—The photolysis products were separated by preparative TLC using
to a potentially embryotoxic free radical intermediate. 30:70 ethyl acetate/hexane as the eluting solvent. Authentic samples of
EXPERIMENTAL PROCEDURES some products were synthesized or purchased and co-eluted to confirm
the identity of products. The separated products were then scraped off
Materials the TLC plate and analyzed by HPLC in line with a tandem mass
Purified PHS-1 and 8-hydroxy-29-deoxyguanosine were obtained spectrometer (HPLC-MS/MS).
from Cayman Chemicals Co. (Ann Arbor, MI); phenytoin (diphenylhy- Identification of Photolysis Products by HPLC-MS/MS—The reac-
dantoin acid), 5-(p-hydroxyphenyl)-5-diphenylhydantoin (HPPH), tion mixture of each time interval as well as the separated products
dimethadione, a-phenyl-N-t-butylnitrone (PBN), hematin, hydroxyla- obtained from TLC studies were analyzed by HPLC-MS/MS (API II,
 Free Radical Intermediates of Teratogens 25081

FIG. 2. Postulated bioactivation of

phenytoin and related proteratogens
to an embryotoxic free radical inter-
mediate by embryonic enzymes with
peroxidase activity. Cyclooxygenase
and hydroperoxidase are the components
of PHS. Arachidonic acid released from
membrane phospholipids by phospho-
lipase A2 serves as the co-substrate in
boththecyclooxygenase-andlipoxygenase-
dependent eicosanoid pathways, generat-
ing the corresponding hydroperoxides,
which can then be reduced by hydroper-
oxidases to the corresponding alcohols. In
this pathway, xenobiotics such as phenyt-
oin can serve as the reducing co-sub-
strate, itself being oxidized to a reactive
free radical intermediate. If this free rad-
ical is not detoxified, it can initiate oxida-

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tive stress and/or covalently bind to cellu-
lar macromolecules, thereby causing
irreversible damage and teratogenesis.
PGG2, prostaglandin G2, HPETE, hy-
droperoxyeicosatetraenoic acid; PGH2,
prostaglandin H2; HETE, hydroxyeicosa-
tetraenoic acid. This figure is modified
from Ref. 3.

Perkin-Elmer Sciex, Concord, Canada). The instrument was set in ion in a ST-EPR cavity with a Bruker ER-200 DX band spectrometer. The
spray mode, and the collision activation spectra of the products were instrument settings were as follows: microwave power, 20.5 milliwatts;
obtained using argon as the target gas at an energy of 80 eV. The mean modulation amplitude, 1 G; time constant, 50 ms; scan range, 100 G;
mass 6 S.E. was calculated from the multiply charged ions by the sweep time, 50 s; accumulation, 5 scans; receiver gain, 5.00 3 10 (5);
software Mass spec (version 3.3). The HPLC conditions were the same field center, 3475 G; frequency, 9.81 GHz.
as above. The EPR spectrum for the mixture of the carbon-centered and puta-
Synthesis of Benzophenone Oxime (21)—A mixture of 1 g of benzo- tive nitrogen-centered free radicals was simulated using a standard
phenone, 1 g of hydroxylamine hydrochloride, 5 ml of pyridine, and 5 ml software (ESR 42) developed by Dr. Uwe Oehler (Department of Chem-
of absolute ethanol was heated under reflux for 2 h in a water bath. The istry, University of Guelph, Canada).
solvents were removed by rotoevaporation. The residue was precipi- Oxidation of 29-Deoxyguanosine (29-dG)—29-Deoxyguanosine (1 mg)
tated with 5 ml of ice cold water, and the mixture was vacuum-filtered. was incubated with or without phenytoin in the presence of PHS-1
The oxime was recrystallized from ethanol. Melting point was 142– using the conditions given above with the following modifications: 250
144 °C; HPLC-MS: m/z (MH1 198), 180, 77. mM phenytoin was used, PBN was replaced with 29-deoxyguanosine, and
Bioactivation of Phenytoin and Its Analogs to a Free Radical Reactive 140 mM arachidonic acid was added to start the reaction. The resulting
Intermediate by PHS-1—PHS-1 (1000 units/ml) was incubated with mixture was analyzed by HPLC.
hematin (1.0 mM) and phenol (0.5 mM) for 1 min at 37 °C in 80 mM Detection of 8-Hydroxy-29-deoxyguanosine (8-OH-29-dG)—Oxidation
potassium phosphate buffer, pH 7.9. After addition of the teratogen of 29-dG to 8-OH-29-dG was quantified using an isocratic HPLC system
(500 mM) or its vehicle and the free radical spin trap PBN (1 mM), (Scientific Systems, Inc.) equipped with a 5-mm Spherisorb ODS II C-18
arachidonic acid (AA) (67 mM) was added to start the reaction. After 30 column (15 cm 3 4.6 mm, Jones Chromatography), an electrochemical
min at 37 °C, reactions were terminated and extracted twice with 2 ml detector (model 5100A), a guard cell (model 5020), an analytical cell
of ethyl acetate. The combined ethyl acetate layers were reduced under (model 5010) (Coulochem, ESA, Chelmsford, MA) and an integrator
nitrogen to 500 ml and analyzed by EPR spectrometry. To obtain infor- (Chromapac model CR501, Shimadzu). Samples were eluted using a
mation on a less stable, putative nitrogen-centered radical, phenytoin mobile phase consisting of 50 mM KH2PO4 buffer (pH 5.5) and 5%
also was incubated with PHS-1 for shorter intervals of 2 and 15 min. To methanol at a flow rate of 0.8 ml/min with a detector oxidation potential
block PHS-1-catalyzed bioactivation of phenytoin, the PHS inhibitor of 10.4 V.
ETYA (40 mM) was incubated with the enzyme at 37 °C for 1 min prior Statistical Analysis—Statistical significance of differences between
to the addition of phenytoin and AA. treatment groups was determined by Student’s t test using a standard
The controls for all incubations lacked the respective teratogen but computerized statistical program (Statsview, Abacus Concepts, Inc.).
contained all other components of the incubation including the vehicle The level of significance was p , 0.05.
for the teratogen. For phenytoin and its major in vivo metabolite,
HPPH, saline/NaOH was the vehicle. Trimethadione, dimethadione, RESULTS
and phenobarbital were dissolved in saline. The vehicle for nirvanol,
mephenytoin, and carbamazepine was Me2SO. The concentration of Products of Photochemical Reactions—Over the period of 40
Me2SO in these incubations did not exceed 0.5% (v/v). min, more than 50% of phenytoin was photolyzed to one major
The free radical adducts of PBN were detected at room temperature and three minor products, which were identified by HPLC-
25082 Free Radical Intermediates of Teratogens

FIG. 3. Time course for the genera-

tion of phenytoin photolysis prod-
ucts. Phenytoin (100 mM) was photolyzed
at 300 nm in the presence of 100 mM so-
dium persulfate for up to 40 min. The
products were analyzed by HPLC-MS/MS.

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MS/MS (Fig. 3). The major product of this reaction was 1,2,3,4- TABLE I
tetrahydro-2-oxo-4-phenylquinazoline (1), and the minor prod- Retention times and fragmentation patterns of phenytoin photolysis
products
ucts were benzophenone (2), benzophenone oxime (3), and
Phenytoin was photolyzed in the presence of sodium persulfate. The
1-phenyl-1-[2-hydroxyphenyl]methyl imine (4). The HPLC re- product formation was monitored by HPLC, and the products were
tention times of these compounds are summarized in Table I. identified by tandem mass spectrometry.
The fragmentation pattern for compound 1 (Table I) was
consistent with concomitant loss of a phenyl ring, carbon mon- Retention Molecular Fragmentation
Compound ion
time pattern
oxide, and hydrogen cyanide from this compound. The frag- (MH1)

mentation pattern for compound 2 was consistent with the loss min m/z
of a phenyl ring. Compounds 3 and 4 had the same molecular Phenytoin 4.25 253 225, 182
weight but showed different fragmentation patterns and were 1,2,3,4-Tetrahydro-2-oxo-4- 3.37 223 145, 117, 90, 77
detected at different retention times (Table I). The fragmenta- phenylquinazoline (1)
Benzophenone (2) 19.34 183 78
tion pattern for compound 3 was consistent with a loss of water Benzophenone oxime (3) 12.15 198 180, 77
and phenyl ring, while that of compound 4 was consistent with 1-phenyl-1-[2-hydroxyphenyl]methyl 8.25 198 105, 77
loss of a phenol group and a phenyl ring. imine (4)
The products of the photolysis reactions were separated on
the TLC plate and characterized by HPLC-MS/MS. The frag-
mentation pattern observed for each product was the same as teratogen free radicals by other components, incubations con-
that observed for the reaction mixture. The authentic samples taining all components except the enzyme also were analyzed.
of compounds 2 and 3 had the same RF values by TLC as their Free radicals were not detected in these incubations, indicating
corresponding compounds from the reaction mixture, and their that the two signals at either end of the spectra were due to
MS/MS fragmentation patterns were the same as for their PHS and that free radicals of teratogens were not formed in the
corresponding products of the photolysis reaction. absence of PHS-1 (Fig. 5A).
Bioactivation of Phenytoin and Its Analogs to a Free Radical Time-dependent incubation of phenytoin revealed the early
Reactive Intermediate by PHS-1—PHS-catalyzed formation of simultaneous existence of carbon- and putative nitrogen-cen-
free radical spin adducts were obtained for phenytoin and its in tered free radicals at 15 min (Fig. 5C) and maximally at 2 min
vivo hydroxylated metabolite, HPPH (Fig. 4). The EPR signal (Fig. 5D), with HFSCs of aN 5 13.75 G and abH 5 2.13 G for the
for phenytoin after a 30-min incubation (Fig. 4B) revealed the carbon-centered radical adduct and aN 5 14.2 G, abH 5 0.79 G,
presence of a carbon-centered free radical. The triplet of dou- and abN 5 1.90 G for the nitrogen-centered radical adduct. The
blets observed for this radical adduct of phenytoin had hyper- arrows in Fig. 5D identify the presence of additional lines that
fine splitting constants (HFSCs) of aN 5 13.75 G and abH 5 were formed due to the overlapping signals of the nitrogen- and
2.13 G. HPPH also gave rise to a carbon-centered free radical carbon-centered radical adducts. These additional lines were
(Fig. 4C) with similar HFSCs, aN 5 13.79 G and abH 5 2.38 G. not present in the signal observed for the carbon-centered
Preincubation of PHS-1 with the PHS/lipoxygenase inhibitor radical at 30 min of incubation (Fig. 5B). This characterization
ETYA (40 mM) abolished the free radical EPR signal for phe- was confirmed by computer simulation of the signal (Fig. 5E).
nytoin (Fig. 4D). The 40 mM concentration of ETYA is well The EPR spectra of isomers of mephenytoin and nirvanol
above the Ki value for PHS inhibition in isolated cells and indicated carbon-centered free radicals in varying amounts
purified enzyme preparations (22, 23), is not embryotoxic, and (Fig. 6) with HFSCs similar to those observed for phenytoin
inhibits phenytoin embryotoxicity in embryo culture (24). The (Table II). There was no radical adduct of PBN in the control
control incubation, which contained saline/sodium hydroxide incubations of these compounds (Fig. 6A). We found in our
(Fig. 4A), the vehicle of phenytoin and HPPH, did not show the system that concentrations higher than 0.5% of Me2SO, a rad-
presence of any radical other than the two peaks that are ical scavenger, resulted in elimination of the signal, possibly by
always observed, probably from PHS. inhibiting PHS. We also found that the addition of more than 1
To explore this possibility and that of the generation of mM PBN to incubations resulted in a decrease in intensity of
 Free Radical Intermediates of Teratogens 25083

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FIG. 4. EPR spectra of the vehicle control (A), phenytoin (B),
and HPPH (C) observed after their bioactivation by PHS. The in FIG. 5. Time-dependent incubation of phenytoin with PHS-1.
vitro system contained 1000 units/ml PHS, 1.0 mM hematin, and 0.5 mM Shown are EPR spectra for the incubation of phenytoin without PHS-1
phenol. After preincubation for 1 min at 37 °C, 67 mM AA, the spin trap (A) and for 30- (B), 15- (C), and 2-min (D) incubation of phenytoin with
a-phenyl-N-t-butylnitrone (1 mM) and the teratogen (500 mM) or its PHS-1. E, computer simulation of the 2-min incubation signal. The
vehicle were added, and the system was incubated for 30 min. For HFSCs for the carbon-centered radical adduct were aN 5 13.75 G and
inhibition studies, the PHS inhibitor ETYA (40 mM) (D) was preincu- abH 5 2.13 G, and and for nitrogen-centered radical adduct HFSCs were
bated with PHS for 1 min prior to the addition of phenytoin. The control aN 5 14.2 G, abH 5 0.79 G, and abN 5 1.90 G. The arrows identify the
incubation contained all components, except the drug was excluded presence of additional lines that were formed due to the overlapping
from the vehicle (saline/sodium hydroxide). signals of nitrogen- and carbon-centered radical adducts. Components
of this in vitro system are detailed in the legend to Fig. 4.

dG. Incubations containing phenytoin had a 5.2-fold increase in

the signal, suggesting that higher concentrations of PBN could 29-dG oxidation compared with incubations with the saline
inhibit PHS. vehicle control (Fig. 8).
Both L-mephenytoin and D-mephenytoin were bioactivated
by PHS-1 to carbon-centered free radicals, with a stronger DISCUSSION
signal observed for D-mephenytoin compared with its L-isomer Results obtained from the photolysis of phenytoin in the
(Fig. 6, B and C). The embryotoxic L-isomer of nirvanol pro- presence of a strong oxidizing agent, sodium persulfate, sug-
duced slightly more free radical in the presence of PHS-1 than gest that phenytoin is first oxidized to a nitrogen-centered
the nonteratogenic D-isomer (Fig. 6, D and E). radical a that can rapidly undergo ring opening to form a
Trimethadione and dimethadione were both bioactivated by carbon-centered free radical b with an isocyanate moiety (Fig.
PHS-1 to carbon-centered free radicals (Fig. 7), with HFSCs 9). Radical b and its resonance contributor radical c both react
similar to phenytoin (Table II). Dimethadione is the pharma- with an oxygen molecule to generate compounds 2 and 4. Anal-
cologically active metabolite of trimethadione and, due to its ysis of an authentic sample of compound 4 was not attempted,
longer half-life, accumulates in humans and animals. Separate since imines in which the nitrogen is attached to a hydrogen
administration of these two compounds in pregnant mice sug- are generally unstable and rapidly decompose or polymerize
gests that the teratogenicity of trimethadione results from in (25). Cyclization of radical c produces compound 1. Compound
vivo N-demethylation to dimethadione, which is the penulti- 3 may be formed after oxidation of the decomposition product of
mate teratogenic species (8). This hypothesis is consistent with the radical b. The mechanism postulated in Fig. 9 provides a
the observation in the present study that dimethadione pro- potential chemical basis for the generation and characteristics
duced a strong EPR signal (Fig. 7B), while the less teratogenic of the putative phenytoin free radicals.
parent compound trimethadione produced a very weak signal Bioactivation of phenytoin by the purified PHS-1/AA system
(Fig. 7C) A free radical signal was not detected in control generated a putative nitrogen-centered free radical and a de-
incubations for these two drugs (Fig. 7A). finitive carbon-centered free radical. Inhibition of the phenyt-
Phenobarbital was bioactivated by PHS-1 to a carbon-cen- oin EPR signal by the PHS inhibitor ETYA indicated that the
tered free radical intermediate, which was detected as a weak bioactivation of phenytoin to a free radical intermediate was
EPR signal (Table II). Free radical intermediates were not catalyzed by PHS. ETYA is a dual inhibitor of PHS and lipoxy-
detected in PHS-1 incubations containing the anticonvulsant genases (26 –30) and has been shown in vitro at the same
drug carbamazepine (data not shown). concentration to inhibit both the bioactivation of phenytoin by
In Vitro Phenytoin-initiated Oxidation of 29-dG—Using the PHS and lipoxygenase and the embryotoxicity of phenytoin in
reaction conditions above employed for free radical character- embryo culture, as well as inhibiting phenytoin teratogenicity
ization, phenytoin was bioactivated by PHS-1 in vitro to a free in vivo (24, 31). The absence of an EPR signal when PHS-1 was
radical reactive intermediate that oxidized 29-dG to 8-OH-29- omitted from the medium indicated that bioactivation was due
25084 Free Radical Intermediates of Teratogens

FIG. 6. EPR spectra for vehicle con-

trol (A), L-mephenytoin (B), D-mephe-
nytoin (C), L-nirvanol (D), and D-nir-
vanol (E) observed after their
bioactivation by PHS. Components of
this in vitro system are detailed in the
legend to Fig. 5, with a 30-min incubation.
The control incubation contained all com-
ponents, except the drug was excluded
from the vehicle (0.5% Me2SO).

TABLE II
Relative amounts of free radical detected by ESR and their hyperfine
splitting constants

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The in vitro system contained prostaglandin H synthase, hematin,
and phenol. After preincubation for 1 min at 37 °C, arachidonic acid,
a-phenyl-N-t-butylnitrone, and xenobiotic or vehicle was added, and the
system was incubated for 30 min.
Spectral
Teratogen Hyperfine splitting constants
amplitude

Phenytoin aN 5 13.75, abH 5 2.13

Alone 111 aN 5 14.2, abH 5 0.79, abN 5 1.90
Plus ETYA 1
HPPH 1111 aN 5 13.79, abH 5 2.38
L-Mephenytoin 1 aN 5 13.56, abH 5 2.12
D-Mephenytoin 11 aN 5 13.60, abH 5 2.13
L-Nirvanol 11 aN 5 13.56, abH 5 1.95
D-Nirvanol 11 aN 5 13.66, abH 5 2.12
Trimethadione 1 aN 5 13.64, abH 5 1.97
Dimethadione 1111 aN 5 13.89, abH 5 2.26 FIG. 8. Formation of 8-hydroxy-2*-deoxyguanosine (8-OH-2*-
Phenobarbital 1 aN 5 13.95, abH 5 2.55 dG) during the bioactivation of phenytoin by PHS-1. Incubations
consisted of 29-dG, phenytoin or its vehicle, PHS-1, hematin, and AA as
described in the legend to Fig. 5. The mixtures were incubated for 30
min and analyzed by high performance liquid chromatography with
electrochemical detection. The asterisk indicates a difference from con-
trol (p , 0.05).

around the radical center, as well as a nitrogen or an oxygen

attached to them. For some but not all of these compounds,
there was an interesting correlation between their reported
teratogenicity and the amount of free radical intermediate
formed via the PHS bioactivating system. For instance, the
highly teratogenic dimethadione (8) produced substantially
more free radicals than its minimally teratogenic parent com-
pound trimethadione. This substantial free radical formation
from dimethadione, together with its accumulation during
chronic therapy with its rapidly N-demethylated parent com-
FIG. 7. EPR spectra for vehicle control (A), trimethadione (B), pound trimethadione, probably account for the teratogenicity
and dimethadione (C) after their bioactivation by PHS. Compo- observed with trimethadione therapy during pregnancy. This is
nents of this in vitro system are detailed in the legend to Fig. 5, with a
30-min incubation. The control incubation contained all components, in agreement with the substantial in vitro oxidation of DNA
except the drug was excluded from the vehicle (saline). initiated by dimethadione, compared with none by trimethadi-
one, using a horseradish peroxidase bioactivating system (32).
to PHS rather than other components of the system. The ab- Similarly, phenytoin and its major parahydroxylated metabo-
sence of a signal in the control incubations also confirms that lite HPPH produced similar amounts of free radicals. While
signals observed in each spectrum were the result of bioactiva- HPPH has been reported to be nonteratogenic following mater-
tion of the drug by PHS-1. nal administration, this probably is due to maternal glucu-
Carbon-centered free radicals also were observed in varying ronidation preventing HPPH from reaching the embryo (3– 6,
amounts for the analogs of phenytoin (Table II). The observed 9), since in embryo culture, phenytoin and HPPH demonstrate
similarity between the HFSCs observed for the carbon-cen- similar embryotoxic potencies (34). Phenobarbital produced
tered free radical of phenytoin and its structurally related only minimal free radical formation, while no free radicals were
analogs is due to the similarities in the environment of the detected with carbamazepine, and these drugs are believed to
radical center. All observed carbon-centered radicals in this be less teratogenic in humans (34 –37) and animals (38, 39)
study have either two phenyl, one phenyl, or two methyl groups than phenytoin and trimethadione.
 Free Radical Intermediates of Teratogens 25085

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FIG. 9. Postulated mechanism for the formation of products from phenytoin photolysis in presence of sodium persulfate.

On the other hand, both D- and L-isomers of mephenytoin isomer disposition and embryonic bioactivation may play im-
were bioactivated to carbon-centered free radicals, and the portant roles in teratologic potency.
amount of free radicals formed was higher for the D-isomer, In the cells of normal untreated animals, there is consider-
which is less embryotoxic in mice (7). Furthermore, the amount able oxidative damage (42). However, excessive oxidative DNA
of free radicals formed by D-mephenytoin and both the D- and damage caused by xenobiotic-initiated ROS can cause irrevers-
L-isomers of its N-demethylated metabolite nirvanol were sim- ible modifications to DNA (43). These modifications to DNA
ilar, although L-nirvanol is substantially more embryotoxic have been shown to disrupt transcription, translation, and
than either mephenytoin or D-nirvanol. These results suggest DNA replication, which can ultimately lead to mutation and
that factors in addition to PHS-catalyzed bioactivation to a free cell death (44 – 46). Oxidative damage to embryonic cellular
radical may contribute to the relative teratologic potencies of macromolecules (DNA, protein, lipid) also may play an impor-
mephenytoin and nirvanol isomers. Mephenytoin is adminis- tant role in the mechanism of embryotoxicity for a number of
tered as a racemic mixture, and in humans, the D-isomer of proteratogens (3– 6, 9, 47, 48). The potential teratologic role of
mephenytoin is preferentially and rapidly hydroxylated and damage to DNA in particular is suggested by a number of lines
excreted, with virtually no D-nirvanol being produced via N- of evidence (3– 6, 9), including particularly 1) the oxidation of
demethylation, while the L-isomer of mephenytoin is stereospe- embryonic DNA in embryo culture by proteratogens like phe-
cifically N-demethylated to L-nirvanol (40, 41). A similar stere- nytoin and benzo[a]pyrene (3, 5); 2) the abolition of embryonic
oselective elimination is observed in mice (7). This rapid DNA oxidation and embryotoxicity for both of these proterato-
elimination of D-mephenytoin via maternal hydroxylation may gens by the addition of the antioxidative enzymes superoxide
prevent its transport to the embryo, where it can be bioacti- dismutase or catalase to the culture medium; and 3) the en-
vated by PHS. In turn, while D-nirvanol produced slightly more hanced in vivo teratogenicity of both these proteratogens in
free radicals than the highly embryotoxic L-isomer, most D- knockout mice deficient in the p53 tumor suppressor gene,
mephenytoin administered as an anticonvulsant is hydroxy- which facilitates DNA repair (49 –51). Generally, hydroxyl rad-
lated and excreted, with very little being N-demethylated to ical (zOH) generated chemically or by ionizing radiation can add
D-nirvanol (7). Furthermore, D-nirvanol itself is hydroxylated across the double bonds of a DNA base, forming a hydroxylated
and excreted more rapidly than its L-isomer, leaving less to product. The oxidized guanine analog 8-OH-29-dG is thought to
reach the embryo (7). Thus, for several reasons, little of the be formed in DNA via hydroxylation of deoxyguanosine resi-
D-isomers of either mephenytoin or nirvanol should reach the dues by zOH at the C-8 position (52), and phenytoin has been
embryo. Nevertheless, in an in vitro horseradish peroxidase shown to initiate the in vivo formation of zOH, measured by
bioactivation system, the highly embryotoxic L-nirvanol pro- salicylate hydroxylation (12). Accordingly, 8-OH-29-dG forma-
duced substantial DNA oxidation, compared with minimal tion can be used as a biological marker of oxidative DNA
oxidation by L-mephenytoin or D-nirvanol (32), so both drug damage, as well as providing an insight into potential molecu-
25086 Free Radical Intermediates of Teratogens

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FIG. 10. Postulated role of peroxidases and NADH in the formation of ROS during the bioactivation of phenytoin by PHS-1 to a
free radical reactive intermediate.

FIG. 11. Postulated role of a Fenton-

like mechanism for the generation of
ROS resulting from the bioactivation
of phenytoin by PHS-1 to a free radi-
cal. The phenytoin hydroperoxide is pos-
tulated to form via a carbon-centered free
radical intermediate as shown in Fig. 10.
1, Fenton-like pathway, wherein hydroxyl
radical is generated during the reduction
of phenytoin hydroperoxide by Fe21 to an
alcohol. 2, pathway for generation of su-
peroxide anion, whereby hydroxyl radical
reduces phenytoin hydroperoxide to an al-
cohol and produces superoxide anion,
which subsequently regenerates hydroxyl
radical.
 Free Radical Intermediates of Teratogens 25087
lar mechanisms of toxicological initiation. In the current study, In summary, the radical spin trapped adducts of hydantoins
under in vitro conditions similar to those used for the formation and related proteratogens are formed via PHS-catalyzed bioac-
and characterization of teratogen free radical intermediates, tivation in varying amounts and with similar HFSCs, consist-
arachidonate-dependent, PHS-catalyzed bioactivation of phe- ent with a common mechanism of teratogenesis. In some cases
nytoin resulted in over a 5-fold increase in the oxidation of (phenytoin, HPPH, trimethadione, dimethadione, phenobarbi-
29-dG to 8-OH-29-dG. These results suggest that the free radi- tal, carbamazepine), the amount of free radicals formed corre-
cal intermediates characterized herein for phenytoin and re- lated well with the teratologic potency of the drugs, while for
lated proteratogens are relevant to their molecular mechanism other drugs (mephenytoin and nirvanol isomers), additional
of teratologic initiation, which may involve oxidative damage to factors appeared to be involved. The free radical detected for all
embryonic DNA. hydantoins and related compounds was carbon-centered, and
Based on the results of these studies, at least two biochem- for phenytoin, a putative, unstable nitrogen-centered radical
ical pathways, summarized in Figs. 10 and 11, could account was also detected. This study provides the first direct chemical
for the generation of O2. and other ROS during the bioactivation evidence for PHS-catalyzed bioactivation of phenytoin and re-
of phenytoin by PHS-1. In both pathways, first a nitrogen- lated proteratogens to free radical intermediates that can ini-
centered free radical of phenytoin (a) is generated by PHS-1. tiate DNA oxidation, which may constitute a common molecu-
This nitrogen-centered radical is most likely unstable, since a lar mechanism of teratologic initiation.
shorter incubation time (2 min) was required for its detection Acknowledgments—We thank Dr. Jack P. Uetrecht (Faculty of Phar-
by EPR, and undergoes ring opening to generate the isocya- macy, University of Toronto) for suggesting the structure of compound
nate-containing carbon-centered radical (b). The equilibrium 1; Dr. A. Jerry Kresge (Department of Chemistry, University of
between radicals a and b is probably shifted toward radical b, Toronto) for a review of our postulated photochemical pathway; Dr.
Robert A. McClelland (Department of Chemistry, University of
since the radical a (nitrogen-centered) was observed in a very Toronto) for providing the Rayonet chamber for the photolysis reac-
low concentration, as was evidenced by the low intensity of the

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tions; Dr. Edward Janzen and Dr. D. Larry Haire (Department of
EPR signal. The nitrogen- and carbon-centered radicals of phe- Clinical Studies, University of Guelph) for discussions concerning the
nytoin can be reduced to metabolite c by hydrogen abstraction analysis of free radical intermediates; and Dr. Janzen for the use of the
EPR spectrometer.
from DNA, lipids, and GSH. This process generates DNA
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 Free Radical Intermediates of Phenytoin and Related Teratogens:
PROSTAGLANDIN H SYNTHASE-CATALYZED BIOACTIVATION,
ELECTRON PARAMAGNETIC RESONANCE SPECTROMETRY, AND
PHOTOCHEMICAL PRODUCT ANALYSIS
Toufan Parman, Guoman Chen and Peter G. Wells
J. Biol. Chem. 1998, 273:25079-25088.
doi: 10.1074/jbc.273.39.25079

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