Introduction

Abstract Introduction Results Discussion References

To reduce the risk of harmful seizures to both the mother and child, pharmacological intervention in the treatment of epilepsy is often continued throughout pregnancy. However, as a therapeutic class, antiepileptic drugs are associated with an enhanced risk of birth defects. Although CBZ¹ monotherapy is commonly prescribed to the pregnant epileptic mother (1, 2), CBZ-related teratogenic effects have been observed clinically (3-5). Moreover, the sometimes unavoidable coadministration of other anticonvulsants with CBZ in some individuals has been associated with an increased incidence of birth defects among neonates exposed in utero (3, 6, 7). Lindhout and coworkers (3) reported that prenatal exposure to the combination of CBZ, PB, and valproate was associated with a 58% incidence of congenital anomalies (3). This high rate of malformation could not be explained by the additive toxicities of these three anticonvulsants or simply by the number of anticonvulsants coadministered, because other combinations of 3-4 drugs had a 7% incidence of malformation. On the basis of these findings, and an understanding of the complexities of pharmacokinetic interactions that occur between CBZ and other coadministered anticonvulsants, several investigators have speculated that the teratogenic effects associated with CBZ exposure are due to a metabolite of CBZ rather than the parent drug itself (3, 4, 8, 9).

The metabolic pattern of CBZ is varied and complex. In addition to multiple pathways of elimination that lead to >30 different metabolites, chronic CBZ treatment also increases its own hepatic biotransformation (10). In humans, ~70% of the dose is excreted in urine, and only 2% is excreted as unchanged drug. The biotransformation of CBZ is characterized by four proximate pathways of elimination (11). The first step of the major pathway is oxidative formation of CBZE. CBZE is largely eliminated by hydrolysis to CBZ-10,11-trans-dihydrodiol (in species other than mouse) and conjugation to form O-glucuronides. CBZE has also been postulated as an intermediate in the formation of substituted acridine metabolites from CBZ. In addition, this pathway may also involve minor CBZ metabolites formed by aromatic hydroxylations of CBZE (12). The remaining three pathways of CBZ elimination include: N-glucuronidation, aromatic hydroxylation, and thioether formation. Aromatic hydroxylation and thioether formation of CBZ (or CBZE) can involve the formation of reactive electrophiles.

To study the underlying mechanism responsible for this drug-induced toxicity, a mouse model of CBZ teratogenicity was developed (13). The model was characterized by an increased incidence of fetal malformations observed when PB was coadministered with an otherwise nonteratogenic dose of CBZ. In addition, STP, a broad spectrum inhibitor of cytochrome P450s (14), protected against the teratogenic effects of CBZ when coadministered with a teratogenic dose of CBZ.

The aim of the present study was to identify pathways of CBZ metabolism that could lead to the formation of teratogenic CBZ metabolites in the SWV mouse through the administration of ¹⁴C-CBZ. First, the disposition of ¹⁴C-CBZ was characterized by mass balance to determine (by ¹⁴C-CO₂ generation) whether acridine metabolites of CBZ (15) are extensively formed in the SWV mouse, thus resulting in loss of the label. Second, CBZ metabolites whose formation was modulated by PB and STP cotreatments were identified by changes in the fraction of CBZ dose metabolized to individual urinary metabolites. Given the complexity of CBZ metabolism, metabolites whose formation was modulated in a manner consistent with the results of teratogenicity studies were given high priority for identification, although it was recognized that changes in the qualitative profile of specific CBZ urinary metabolites excreted in urine would not necessarily reflect the true magnitude of cotreatment effects. Thus, this investigation was an initial attempt to identify possible metabolites that could be involved in a mechanism of CBZ teratogenicity.

Materials and Methods

Chemicals. ¹⁴C-CBZ, labeled at the carbonyl carbon of the side chain (10 mCi/mmol), was purchased from Sigma Chemical Co. (St. Louis, MO). The chemical and radiochemical purities were >99% by HPLC. ¹⁴C-CBZ was diluted with unlabeled CBZ (Abbott Laboratories, Chicago, IL) to a final specific activity of 2.25 mCi/mmol. 2-Hydroxy CBZ and 3-hydroxy CBZ were the gifts of Dr. René Levy (University of Washington, Seattle, WA). STP was supplied by Biocodex Research Laboratories (Montrouge, France). CBZ-10,11-trans-dihydrodiol was prepared as described previously (16) from CBZE that was synthesized by the method of Bennett et al. (17). 45% Aqueous 2-hydroxypropyl--cyclodextrin was obtained from Research Biochemicals International (Natick, MA); PB and -glucuronidase (EC 3.2.1.31) crude solution were obtained from Helix pomatia; hydrogen peroxide was obtained from Sigma; ethanolamine and 2-ethoxyethanol were obtained from Aldrich Chemical Co. (Milwaukee, WI); and trifluoroacetic acid was obtained from Pierce Chemical Co. (Rockford, IL). L-Ascorbic acid and Optima grade methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA); ammonium acetate, barium hydroxide, and hydrochloric acid were purchased from J. T. Baker, Inc. (Phillipsburg, NJ); Ready-Safe liquid scintillation cocktail was purchased from Beckman Instruments, Inc. (Fullerton, CA); and Flo-Scint II LSC cocktail was purchased from Packard (Downers Grove, IL).

Animals. The highly inbred SWV/Fnn mouse strain used in this study has previously been developed as a model of CBZ-related teratogenicity (13). This mouse colony was housed and maintained according to approved animal care practices at Texas A & M University. Mature female SWV/Fnn mice (20-25 g) were used for all biological experiments.

CBZ Urinary Metabolites. Mice were randomly assigned to three treatment groups: CBZ, 1,500 mg/kg/day; CBZ, 1,500/STP 300 mg/kg/day; and CBZ, 1,000/PB, 45 mg/kg/day. The dose of STP (300 mg/kg/day) was previously determined to be protective against a teratogenic dose of CBZ (1,500 mg/kg/day), whereas the coadministration of PB (45 mg/kg/day) had been found to produce teratogenicity with a nonteratogenic dose of CBZ (1,000 mg/kg/day) in the SWV mouse (13). During the 7-day pretreatment period, mice were housed in polyethylene cages with sawdust bedding. Food and water were provided ad libitum. CBZ was dosed alone in food or in combination with either PB (by gavage) or STP (in food). On day 7, ¹⁴C-CBZ (8 mg/kg, 5 µCi) dissolved in 45% aqueous 2-hydroxypropyl--cyclodextrin was administered intraperitoneally (0.1 ml/mouse). Mice were grouped by regimen and placed into polyethylene metabolic cages (5 mice/cage) (Nalgene Co., Rochester, NY) for 24 hr and received water ad libitum, but no food. Urine was collected over ascorbic acid (2 mg) and stored at 70°C until analysis.

Mass Balance of Radiolabel. Mice (N = 5) received CBZ (20 mg/kg/day) in cyclodextrin intraperitoneally over a 7-day pretreatment period. Food and water were provided ad libitum. On day 7, ¹⁴C-CBZ (8 mg/kg, 5 µCi) was administered by ip injection, as described previously. The actual injected dose was determined gravimetrically. After administration of ¹⁴C-CBZ, mice were placed into a single sealed glass metabolic cage (Lab Glass, Inc., Vineland, NJ) for collection of excreta and ¹⁴CO₂ (18, 19). Water was provided ad libitum. Positive air flow through the glass cage was maintained by connection to an aspirator. Air was drawn through a lime trap in series before the cage, and expired air was bubbled through three liquid reservoirs connected in series between the cage and the aspirator. The reservoirs were arranged such that expired air was first bubbled through aqueous HCl (4% v/v) before going to the first CO₂ trap, which contained an ethanolamine-ethoxyethanol mixture (1:2). The second CO₂ trap contained a saturated BaOH solution (20% w/v). The ethanolamine-ethoxyethanol mixture was replaced at 8 and 16 hr and the BaOH solution at 12 hr. The animals were euthanized under an atmosphere of ether 24 hr after administration of ¹⁴C-CBZ. Tissues and excreta were collected at 24 hr for the determination of ¹⁴C disposition. Cage parts were rinsed with methanol until counts declined to background levels. The methanol rinse was collected, reduced (<4 ml) by rotary evaporation, and added to the pooled urine sample from which a small aliquot was removed to determine the concentration of radiolabel by LSC. Feces were added to a solution of sodium methoxide (2 M) and repeatedly vortexed to achieve a slurried mixture. Before LSC, an aliquot of the slurry was decolorized with hydrogen peroxide (30%) and adjusted to a neutral pH with concentrated HCl. The viscera were removed from the carcasses and homogenized mechanically in sodium methoxide (2 M). Carcasses were cut into small pieces after removing the skin and homogenized mechanically in sodium methoxide (2 M). A small aliquot of each homogenate mixture was sampled for the quantitation of radiolabel by LSC. Exhaled ¹⁴CO₂ contained in the BaOH solutions (0.5 ml diluted with 0.5 ml H₂O) and ethanolamine-ethoxyethanol solutions (1 ml) were also determined by LSC.

Chromatographic and Spectroscopic Analysis of CBZ Metabolites. Three HPLC systems are described. The isolation of minor CBZ metabolites for which no synthetic standards exist was necessary before mass spectral analysis. In general, urinary CBZ metabolites could not be resolved using mobile phase conditions optimized for atmospheric pressure ionization.

System 1. Neat and hydrolyzed urine was analyzed using a Hewlett-Packard Series 1050 HPLC with a variable-wavelength UV detector set at 230 nm. For some analyses, an A-100 -radioactive flow detector (Radiomatic, Tampa, FL) with Flo-Scint II LSC cocktail was arranged in series after the UV detector. Absorbance spectra of specific chromatographic peaks were obtained using a Hewlett-Packard Series II 1090 HPLC with a diode array detector scanning from 200 to 350 nm. These HPLC systems were equipped with a 5 µm Econosil C₈ column (25 cm × 4.6 mm; Alltech Associates) protected by a C₈ guard column (10 mm × 4.6 mm; Alltech Associates). The mobile phase consisted of solvent A (10 mM ammonium acetate, pH 7) and solvent B (50% methanol and 50% acetonitrile) delivered at a constant flow rate of 1 ml min¹. The gradient mixture was initially held at 10% B (0-10 min), increased linearly to 35% B (10-35 min), maintained at 35% B (35-45 min), and increased linearly to 55% B (45-65 min). Retention times of the synthetic standards were: CBZ-10,11-dihydrodiol, 32 min; 2-hydroxy-CBZ, 41 min; CBZE, 42 min; 3-hydroxy-CBZ, 43 min; and CBZ, 52 min.

System 2. CBZ urinary metabolites were partially purified using a Beckman 112 Solvent Delivery Module and a 5 µm Ultremex C₁₈ column (25 cm × 10 mm; Phenomenex, Torrance, CA) protected by a C₁₈ guard column. UV absorbance was monitored using a Hewlett-Packard Series 1050 detector set at 230 nm. Mobile phase mixtures of acetonitrile (10-35%) in 10 mM ammonium acetate were isocratically delivered at a constant flow rate of 3 ml min¹. The proportion of acetonitrile in the mobile phase was adjusted for the relative polarity of the metabolite being isolated. Fractions were collected by hand or using a fraction collector (Frac-100, Pharmacia, Sweden). In some cases, individual fractions were further purified using system 1. Fraction eluents collected using system 1 or system 2 were frozen, lyophilized, and stored at 20°C. Lyophilized samples were reconstituted in ammonium acetate buffer (0.01 M, pH 7) to achieve a 2- to 5-fold concentrated mixture (~100 µl) just before mass spectral analysis.

System 3. On-line LC/MS and LC/MS/MS of partially purified metabolites were conducted with a Shimadzu LC-10AD twin-pump system equipped with a 5 µm Solvent Miser C₈ column (25 cm × 2.1 mm; Alltech Associates) and a Shimadzu SPD-10AV variable wavelength detector (220 nm) positioned in series just before the mass spectrometer. Isocratic mixtures of solvent A (0.05% trifluoroacetic acid, v/v) and solvent B (50% methanol:50% acetonitrile) ranging from 35 to 55% B were delivered at a constant rate of 0.2 ml min¹. The total HPLC effluent was introduced to the mass spectrometer at a rate of 0.2 ml min¹ without the use of a splitting tee.

MS. Positive-ion mass spectrometric analyses were performed on a VG Quattro II (Fisons Instruments, Manchester, UK) triple quadrupole instrument equipped with an ESI interface. Nitrogen was maintained at constant flow rates for use as the drying (500 liters/hr) and nebulizer (20 liters/hr) gases. The ionization source was heated to 125°C and a potential of 3.8 kV was applied to the ESI probe tip. Cone voltages ranging from 20 to 30 V were selected to optimize the ionization of each analyte. MS/MS was based on CID using argon as the collision gas at a pressure of 2.00 × 10³ mbar (1 mbar = 100.004 Pa). Selected ions entering the radiofrequency-only quadrupole region were subjected to low collision offset energy (20 eV). To obtain product ion and precursor ion spectra, the mass spectrometer was scanned (2 sec) from m/z 50 to m/z 800.

	Results

Abstract Introduction Results Discussion References

CBZ Mass Balance. The distribution of radiolabeled material (0-24 hr collection) in CBZ-pretreated mice after intraperitoneal administration of ¹⁴C-CBZ (8 mg/kg, 5 µCi) is shown in fig. 1. Nearly 68% of the radioactive dose was excreted in urine and feces, consistent with previous findings in humans and rats (11, 12). A preliminary study determined that ~50% of a single CBZ dose in mice was excreted in urine from 0 to 12 hr, 7% from 12 to 24 hr, and 1% from 24 to 48 hr. The amount of radiolabel remaining in the viscera and carcass at 24 hr accounted for <22% of the dose. Approximately 2% of the dose was exhaled in the form of ¹⁴CO₂. Less than 8% of the dose was unaccounted for. These findings indicate that a significant proportion of a CBZ dose is excreted in urine within 24 hr. Moreover, CBZ metabolism involving loss of the ¹⁴C-containing carbamoyl side chain does not constitute a significant pathway of elimination in the SWV mouse model. On the basis of these findings, we focused on the identification of CBZ metabolites in the urine of pretreated SWV female mice.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Mass balance recovery of 14C-radiolabel in the SWV mouse. 14C-CBZ (8 mg/kg, 5 µCi) dosed to 5 female mice after a 7-day CBZ (20 mg/kg/day) pretreatment period. Distribution of radiolabel determined at 24 hr in expired air, viscera, carcass, and excreta. Total recovery was 92.5% of dose.

Effect of Pretreatments on CBZ Urinary Metabolites. To contrast the effects of PB and STP on CBZ metabolism, pooled urine from treated animals was analyzed by HPLC and MS. Female mice pretreated (7 days) with CBZ in the absence and presence of PB or STP received a single ¹⁴C-CBZ dose (8 mg/kg, 5 µCi) by ip injection. CBZ metabolites were separated by HPLC using chromatographic system 1 and analyzed by UV and radiometric detection. Urine from each exposure group was incubated alone or with -glucuronidase added. A comparison of urine samples before and after -glucuronidase treatment revealed that 25-33% of ¹⁴C-CBZ derived material was hydrolyzed (chromatograms not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of drug pretreatment on CBZ urinary chromatographic profiles. HPLC radiometric analyses of urine, hydrolyzed with -glucuronidase (37°C, pH 4.6, 18-20 hr), collected (0-24 hr) from CBZ 1,000/PB 45 mg/kg/day (a), CBZ 1,500 mg/kg/day (b), and CBZ 1,500/STP 300 mg/kg/day (c)-treated SWV female mice. HPLC separations of CBZ metabolites using chromatographic system 1.

Identification of CBZ Metabolites (Peaks A-J). Synthetic standards coinjected individually with hydrolyzed urine confirmed the identity of peaks F, G, H, and J as 2-hydroxy-CBZ, CBZE, 3-hydroxy-CBZ, and CBZ, respectively. The 10,11-dihydrodiol-CBZ standard did not coelute with any of the 10 radioactive peaks, consistent with the low microsomal epoxide hydrolase activity in the SWV mouse (20, 21). Characterization of the remaining CBZ urinary metabolites (described in descending order of urinary abundance) was achieved by spectroscopic analyses, because synthetic standards were not available.

Peak B and Peak C. Metabolites B and C (20 min and 25 min, respectively; fig. 2b) collectively accounted for 19-37% of the total urinary radioactivity of each treatment group after -glucuronidase hydrolysis. Each of these metabolites was successfully isolated from hydrolyzed urine by semipreparative HPLC using chromatographic system 2.

Peak A. Isolation of metabolite A (12 min; fig. 2b) from hydrolyzed samples of pooled urine was accomplished by semipreparative HPLC using chromatographic system 2. Full-scan LC/MS of the collected peak A revealed a protonated molecular ion at m/z 416 (as shown in table 2). Abundant ion fragments at m/z 253 (base peak) and m/z 237 were also evident. These ions are commonly observed in the mass spectra of CBZ metabolites and respectively correspond to an oxidized CBZ moiety and CBZ itself. The assignment of m/z 416 as the molecular ion of peak A, for which no synthetic standard existed, was based on the presence of a sodium adduct at m/z 438 and was further supported by LC/MS/MS in which a precursor ion scan of m/z 253 revealed a single peak at m/z 416 (spectrum not shown). The even-mass molecular ion (m/z 416) suggested a CBZ metabolite with a conjugate group that contained nitrogen (26). Thus, the mass spectral data of metabolite A are most consistent with a NAC conjugate of an oxidized metabolite of CBZ. Additional structural information regarding peak A was obtained using a diode array detector with chromatographic system 1. The spectrum contained absorbance maxima at ~220 and 280 nm. The absorbance maximum near 280 nm is diagnostic of a double bond joining carbons 10 and 11 of the CBZ moiety, and thus suggested that the hydroxyl and NAC substituents are positioned on a nonaromatic 6-membered ring of CBZ.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Metabolite A, dihydrohydroxy-CBZ-NAC. Product ion spectrum of m/z 416 and proposed structure. The origins of structurally diagnostic product ions at m/z 253 and m/z 269 are shown, and the identities of other product ions are described in the text.

Peak D and Peak E. The urinary excretion of metabolites D and E (34 min and 36 min, respectively; fig. 2b) each accounted for ~1% of the administered CBZ dose (table 1). Relatively low urinary abundance and incomplete chromatographic separation of these metabolites limited the ability to make quantitative comparisons between treatment groups. However, using chromatographic system 3, it was possible to achieve complete resolution of metabolites D and E (8 min and 13 min) under isocratic conditions (35% B), while monitoring at 220 nm (chromatograms not shown). To measure the effects of PB and STP on the excretion of these minor CBZ metabolites using system 3, metabolites D and E were partially purified from hydrolyzed urine samples of each treatment group. Chromatographic system 1 was used to collect a single fraction containing metabolites D and E (33-36 min) from each urine sample. Aliquots collected from two injections per sample (18,000 dpm; 3-25 µl) were lyophilized and resolubilized in ammonium acetate buffer (100 µl). Each reconstituted sample (18 µl) was analyzed using chromatographic system 3 and the peak areas (220 nm) of metabolites D and E were compared. The results of these comparisons indicated that PB coadministration was associated with a 5% greater excretion of metabolite D and a 31% greater excretion of metabolite E, relative to the respective amounts excreted by female mice treated with CBZ alone. Conversely, STP coadministration was associated with the diminished excretion of metabolites D and E (9% and 22%, respectively) relative to the CBZ-only treatment group. These results are consistent with the relative changes in metabolites D and E measured as a function of total urinary radioactivity (table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Metabolite D, S-cysteinyl-CBZ. Product ion spectrum of m/z 356 and proposed structure. The origins of structurally diagnostic product ions at m/z 224 and m/z 267 are shown, and the identities of other product ions are described in the text.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Metabolite E, methylsulfonyl-CBZE. Product ion spectrum of m/z 331 and proposed structure. The origins of structurally diagnostic product ions at m/z 179, m/z 209, m/z 258, and m/z 288 are shown, and the identities of other product ions are described in the text.

Peak I. Metabolite I (42 min; fig. 2b) was isolated by HPLC using chromatographic system 1. The presence of 3-hydroxy-CBZ in the fractions was inevitable, because metabolite I was a shoulder peak of the more abundant metabolite H (3-hydroxy-CBZ). Therefore, the full mass spectrum of metabolite I (obtained by LC/MS) was complicated by the presence of predominant ions at m/z 253 (base peak) and m/z 210 that respectively correspond to MH⁺ of 3-hydroxy-CBZ and a protonated 3-hydroxyiminostilbene ion (table 2). Also present in the spectrum were the proposed MH⁺ and sodium adduct of metabolite I appearing at m/z 315 and m/z 337, respectively. An indicated mass of 314 Da was consistent with a postulated elemental composition of C₁₆H₁₄N₂O₃S that suggested metabolite I was a methylsulfonyl adduct of CBZ, a CBZ metabolite previously identified in rat urine by Lertratanangkoon and Horning (12). Furthermore, reanalysis of hydrolyzed urine samples at a higher wavelength (280 nm) indicated the presence of a peak corresponding to metabolite I at 42 min (chromatographic system 1). These data were consistent with absorbance maxima at 220 and 280 nm associated with metabolite I and suggested the methylsulfonyl substituent was attached to an aromatic ring of CBZ. A product ion scan of metabolite I obtained by CID of MH⁺ (m/z 315) is shown in table 3. Class-characteristic cleavage of the carbamoyl side chain afforded an oxocarbonium product ion (m/z 298), protonated (m/z 272) and deprotonated (m/z 270) methylsulfonyl-iminostilbene ions, and a protonated iminostilbene product ion at m/z 193. On the basis of these findings, the identity of metabolite I was most consistent with methylsulfonyl-CBZ.

	Discussion

Abstract Introduction Results Discussion References

We have characterized the pathways of CBZ elimination in the SWV mouse model of CBZ teratogenicity and have identified metabolites that are likely to contribute to the teratogenicity observed with the drug. Initial studies demonstrated that the position of the radiolabel used to identify metabolites relevant to teratogenicity was not lost in vivo.

Approximately 75% of the total urinary radioactivity was identified after enzymatic hydrolysis using a combination of spectroscopic techniques. 3-Hydroxy-CBZ and glucuronides of CBZ and CBZE accounted for the majority of CBZ metabolism in the SWV mouse. Several other previously identified CBZ minor metabolites were also found, including 2-hydroxy-CBZ, CBZE, and methylsulfonyl-CBZ. In addition, two novel CBZ metabolites were tentatively identified, S-cysteinyl-CBZ and dihydrohydroxy-CBZ-NAC, as well as one formed from CBZE, methylsulfonyl-CBZE.

A proposed scheme of CBZ metabolism in the SWV mouse is shown in fig. 6. The primary pathways of CBZ elimination included: (i) N-glucuronidation of CBZ, (ii) epoxidation of CBZ with (iii) subsequent N-glucuronide formation, (iv) ring-oxidations to form CBZ phenols, and (v) phenol conjugation with glucuronic acid. In addition, a series of minor pathways exist (vi) and (vii) that share common intermediate steps in the formation of CBZ thioether metabolites. In addition to results presented herein, the suggestion that phenol formation involves arene oxide intermediates is based on the identification of CBZ phenols, catechols, dihydrodiols and thioether metabolites (11, 12), the recent identification of GSH conjugates in rat bile (25), and covalent binding studies in mouse liver microsomes by us (30) and others (31, 32).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Proposed scheme of CBZ metabolism in the SWV mouse. The interrelationships of multiple pathways of CBZ elimination, reactive intermediate formation, and identities of metabolites corresponding to peaks A-J are shown.

The mercapturic acid and CYS--lyase pathways of GSH adduct catabolism in the context of CBZ elimination are shown in fig. 7. A general scheme is depicted to illustrate the possible formation of the CBZ thioether metabolites A, E, D, and I by a common mechanism, as well as to acknowledge the possibility of alternate metabolic routes. The nonenzymatic dehydration of dihydrohydroxy-S-glutathionyl adducts of CBZ or CBZE (xi) yields the respective S-glutathionyl conjugates that may undergo further catabolism. Alternatively, the dihydrohydroxy-S-glutathionyl conjugates may be metabolized directly by enzymes of the CYS--lyase pathway. Similar biotransformations of the dihydrohydroxy-S-glutathionyl adducts of naphthalene (33) and bromobenzene (34) have been reported. Thus, formation of the respective CYS conjugates may occur by sequential metabolism of the S-glutathionyl or dihydrohydroxy-S-glutathionyl conjugates by -glutamyltranspeptidase (xii) and CYS-glycine transpeptidase (xiii). The acetylation of CYS conjugates is catalyzed by N-acetyltransferase (xiv), whereas soluble deacetylases located in the gastrointestinal tract may catalyze the reverse reaction (xv) (35). Alternatively, CYS conjugates are also substrates for CYS--lyase (xvi) to form a corresponding thiol. Aromatic thiols may be subsequently methylated by thiol-methyltransferases (xvii) and sequentially oxidized to the respective methylsulfinyl (xviii) and methylsulfonyl (xix) conjugates (36-38). In addition, it should be mentioned that nonenzymatic dehydration of the dihydrohydroxy-S-linked adducts could occur at any of the steps (xii-xix); however, for simplicity, only step (xi) is shown.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Proposed scheme of mercapturate and CYS--lyase pathways of CBZ elimination. The identities and relative placements of metabolites A, D, E, and I are shown.

Arene oxide intermediates, that could lead to the formation of thioether conjugates present in urine, have been postulated to account for CBZ-related hypersensitivity reactions (39) and teratogenicity (3, 6). Hypersensitivity and teratogenic effects associated with CBZ exposure may share a common underlying mechanism related to CBZ metabolism. Hypersensitivity is thought to occur secondary to hapten formation by an electrophilic species formed from CBZ and an endogenous protein. Teratogenicity may occur by the binding of a CBZ electrophile to a critical protein essential for normal embryonic development. The extensive body of evidence suggesting that CBZ is eliminated through an arene oxide has lead to the hypothesis that an inherited defect in epoxide hydrolases may account for an unusual susceptibility for hypersensitivity reactions (39), as well as for congenital malformations (40). However, attempts to correlate epoxide hydrolase activity with the incidence of CBZ hypersensitivity have not been successful (41, 42), leading one group of investigators to conclude that patient variability in epoxide hydrolase activity may not account for the observed adverse reactions (41).

Although there is strong evidence that CBZ (and CBZE) forms arene oxide intermediates, it should be acknowledged that arene oxides may not account for the teratogenicity associated with CBZ. Our microsomal studies suggested that CBZ may also form a quinone intermediate (30). Moreover, the identification of thioether metabolites of CBZ and CBZE is consistent with maternally generated metabolites that may be transported to the conceptus, whereas if an arene oxide accounted for the teratogenicity, it may have to be formed by the fetus. The likelihood of transport and teratogenic potential of CBZ thioether metabolites is consistent with the recently reported transplacental transfer, irreversible binding, and developmental toxicity in mice of 3-methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene, a metabolite of the chlorinated insecticide DDT (43).

Comparison of the effects of PB and STP cotreatments on CBZ metabolism in the SWV mouse revealed that the relative urinary excretion of three CBZ metabolites is consistent with the modulation of teratogenicity by these pretreatments as reported previously (13). The relative abundance of dihydrohydroxy-CBZ-NAC,S-cysteinyl-CBZ, and methylsulfonyl-CBZE (peaks A, D, and E, respectively) were increased by PB and decreased by STP coadministrations in a manner consistent with a contribution to teratogenicity by these three metabolites, their precursors, or subsequent products. Of particular interest is the finding that metabolic pathways of both CBZ and CBZE involving the formation of electrophiles are modulated by PB and STP treatments. The identification of methylsulfonyl-CBZE is particularly significant, because we recently provided evidence that formation of CBZE seems to be an important first step in forming the ultimate teratogen in this model (17).