Introduction

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The glucuronidation and elimination of endogenous compounds (e.g., bilirubin) and xenobiotics, including HPPH, the major, para-hydroxylated metabolite of the anticonvulsant drug phenytoin (diphenylhydantoin) (Butler, 1957), are catalyzed by a superfamily of membrane-bound isozymes known collectively as UGTs (Dutton, 1980). UGTs catalyze the conjugation of xenobiotics to UDP-glucuronic acid, allowing the conjugated product to be excreted in the urine and feces. The teratogenicity of phenytoin and related xenobiotics in animals and humans is thought to be due to their bioactivation to embryotoxic reactive intermediates (for reviews, see Hansen, 1991; Juchau et al., 1992; Winn and Wells, 1995a; Wells and Winn, 1996). UGT-catalyzed glucuronidation and elimination may prevent competing bioactivation of such xenobiotics to toxic reactive intermediates that can initiate a spectrum of toxicological sequelae (fig. 1). In animals and humans, UGTs have been shown to be important cytoprotective modulators in 1) B[a]P-initiated micronucleus formation (Vienneau et al., 1995), embryotoxicity (Wells et al., 1989), molecular damage and cytotoxicity (Hu and Wells, 1992, 1993, 1994); 2) micronucleus formation initiated by the tobacco carcinogen NNK (Kim and Wells, 1996a); and 3) in vivo bioactivation (de Morais et al., 1992a,b), hepatotoxicity and nephrotoxicity (de Morais et al., 1992a) of the analgesic drug acetaminophen.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Postulated genoprotective and cytoprotective roles of UGTs in peroxidase-catalyzed phenytoin and HPPH bioactivation and toxicity. Hereditary UGT deficiencies may allow greater bioactivation to free radical reactive intermediates that can initiate the formation of reactive oxygen species. These reactive intermediates and reactive oxygen species can irreversibly damage DNA, proteins and lipids via covalent binding and oxidation, potentially initiating teratogenesis and other toxicities. Similarly, UGTs also may protect against P450-catalyzed bioactivation of phenytoin to an electrophilic, arene oxide, reactive intermediate (not shown), which, particularly postnatally, may result in hepatotoxicity and other toxicities (Winn and Wells, 1995a; Wells and Winn, 1996). The contribution of molecular target oxidation to idiosyncratic drug reactions and reversible lymphoma initiated by phenytoin is speculative. Gluc, glucuronide conjugate; UDPGA, UDP-glucuronic acid; GSH, glutathione.

UGTs exist as two families, UGT1 and UGT2, which are located on separate chromosomes (Moghrabi et al., 1992; Monaghan et al., 1992) and are regulated by distinctly different mechanisms. UGT1 isozymes are produced by alternative splicing of the UGT1 gene complex (Brierly and Burchell, 1993). The UGT1 gene complex exists as multiple isozyme-specific exons that are located at the 5-variable/specific region and that are spliced with a group of four exons at the 3-constant region, the latter being common to all UGT1 isozymes. Conversely, UGT2 isozymes are produced from separate and complete genes located on various chromosomes.

Gilbert's syndrome, a moderate hereditary bilirubin-UGT deficiency due to UGT1*1 gene mutations, is estimated to occur in 6% to 13% of the population (Odell and Childs, 1980; Monaghan et al., 1996). The Crigler Najjar syndromes (types I and II), which are more severe forms of bilirubin-UGT deficiency, have been suggested to occur in 0.1% of the population in a heterozygous form (Bosma et al., 1995). UGT deficiencies in both humans (Bosma et al., 1992; Moghrabi et al., 1993a,b) and rats (Iyanagi, 1991) are due to various mutations in either the variable or constant exon regions. People who have deficient UGTs for catalyzing the glucuronidation and elimination of bilirubin (UGT1*1) phenotypically express abnormally elevated bilirubin blood concentrations and thus appear jaundiced (Moghrabi et al., 1993a,b). Heterozygous and homozygous mutations in other specific UGT exons, such as UGT1*4, have been reported with respective frequencies of 16% and 6% (Burchell et al., 1994).

Hereditary UGT deficiencies in rats and humans have been shown to decrease the glucuronidation of the analgesic drug acetaminophen and the environmental carcinogen/teratogan B[a]P, resulting in enhanced bioactivation, molecular target damage and various toxicities. With acetaminophen, enhanced bioactivation was evident in UGT-deficient humans (de Morais et al., 1992a) and enhanced hepatotoxicity and nephrotoxicity in several strains of UGT-deficient rats (de Morais et al., 1992b). In human lymphocytes, decreased UGT activity for B[a]P metabolites correlated with enhanced cytotoxicity (Hu and Wells, 1993), whereas in vivo and in vitro studies with UGT-deficient rats showed reduced glucuronidation of B[a]P metabolites, resulting in enhanced B[a]P bioactivation, molecular target damage and, in pregnant animals, embryotoxicity (Wells et al., 1989; Hu and Wells, 1992, 1994).

Recent in vitro studies showed that B[a]P- and NNK-initiated micronucleus formation, a form of genotoxicity thought to reflect carcinogenic initiation, was higher in cells cultured from UGT-deficient RHA or Gunn rats, compared with UGT-normal congenic controls (Vienneau et al., 1995; Kim and Wells, 1996a). A similar study in cultured Wistar rat skin fibroblasts, using inducers and inhibitors of both P450s and peroxidases and exogenous addition of superoxide dismutase, suggested that reactive oxygen species-mediated DNA oxidation produced by peroxidase- and/or P450-catalyzed B[a]P bioactivation was a potential molecular mechanism in micronucleus formation (Kim and Wells, 1994, 1995, 1996a), which is thought to reflect the potential for the initiation of cancer and may similarly reflect teratological initiation.

Similarly to B[a]P, a number of studies suggest that both phenytoin and HPPH are bioactivated by peroxidases, such as PHS, to free radical intermediates, the former of which can oxidize lipids, proteins and DNA (Winn and Wells, 1995a; Parman et al., 1996) (fig. 1). Phenytoin also has been shown in vivo to initiate the production of hydroxyl radicals, measured by salicylate hydroxylation (Kim and Wells, 1996b). Although HPPH was reported to be nonteratogenic in pregnant mice after in vivo administration (Harbison and Becker, 1974), this may have been due to maternal glucuronidation preventing HPPH from reaching the embryo. Because UGTs catalyze the glucuronidation and elimination of HPPH (Vore et al., 1979), we hypothesized that UGT deficiencies may increase susceptibility to various phenytoin toxicities via HPPH-initiated genotoxicity, reflected in this study by micronucleus formation. This mechanism might be relevant not only to the teratogenic effects of phenytoin (Winn and Wells, 1995a) but also to other potential consequences of phenytoin genotoxicity (fig. 1). For example, the mechanisms underlying the idiosyncratic drug reactions (fever, rash, etc.) and reversible lymphoma caused by phenytoin (Porter, 1989) have yet to be established.

In this study, we evaluated the potential for UGT-catalyzed genoprotection against phenytoin- and HPPH-initiated micronucleus formation in skin fibroblasts cultured from heterozygous (+/j) and homozygous (j/j) UGT-deficient Gunn rats vs. congenic UGT-normal controls (+/+). Although in vivo metabolism for most pathways occurs primarily in the liver, this in vitro skin fibroblast system has proven useful for characterizing the genoprotective role of UGTs for other teratogens and carcinogens, such as B[a]P (Vienneau et al., 1995) and NNK (Kim and Wells, 1996a). The embryopathic potential and potency of HPPH were determined directly in a mouse embryo culture model that has been well characterized for phenytoin embryopathy (Winn and Wells, 1995a,b) and avoids the confounding effect of maternal glucuronidation. The increased genotoxicity of phenytoin in UGT-deficient cells provides the first evidence that direct N³-glucuronidation of phenytoin, confirmed in this study in vivo by tandem MS, appears to constitute an important and heretofore unrecognized cytoprotective reaction, in addition to the O-glucuronidation anticipated for and observed with HPPH. The direct and potent embryopathic effects of HPPH in mouse embryo culture and the enhanced genotoxicity of HPPH, as well as phenytoin, in even heterozygous UGT-deficient cells suggest that human UGT deficiencies may be important determinants of susceptibility to the toxicity and teratogenicity initiated by phenytoin and related xenobiotics.

	Materials and Methods

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Animals

Male HsdBlu:Gunn rats (180-200 g; Harlan Sprague Dawley Inc., Indianapolis, IN), and age-matched Wistar rats (200-250 g; Charles River Canada Ltd., St. Constant, Quebec), the UGT-normal parent strain of the Gunn rat, were housed in separate plastic cages. Virgin female CD-1 mice (Charles River Canada) were housed in plastic cages with ground corn cob bedding (Beta Chip; Northeastern Products Corp., Warrensburg, NY). Three females were housed with one male breeder from 5:00 P.M. to 9:00 A.M. The presence of a vaginal plug in a female mouse was considered as gestational day 1, and the pregnant females were separated from the colony and housed together in groups of five or fewer animals per cage.

All animals were kept in a temperature-controlled room with a 12-hr light-dark cycle (automatically maintained). Food (Laboratory Rodent Chow 5001; PMI Feeds Inc., St. Louis, MO) and tap water were provided ad libitum. Animals were acclimatized for a minimum of 1 week. All animal studies were approved by the University Animal Care Committee, in accordance with the standards of the Canadian Council on Animal Care.

Chemicals

Phenytoin, HPPH, 4,6-diamidino-2-phenylindole, ribonuclease A, ribonuclease T₁ and Escherichia coli alkaline phosphatase were purchased from Sigma Chemical Co. (St. Louis, MO). 8-OH-2-dG was purchased from Cayman Chemical Co. (Ann Arbor, MI). All other reagents used were of analytical or HPLC grade. Dulbecco's modified Eagle medium, FBS, lyophilized penicillin/streptomycin, HBSS (without calcium chloride, magnesium chloride and magnesium sulfate), Waymouth's MB 752/1 medium, sodium bicarbonate solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, L-glutamine and 0.25% trypsin were purchased from Gibco BRL (Toronto, Ontario).

Cell Culture Studies

The cell culture methods have been described in detail elsewhere (Vienneau et al., 1995).

Cell culture method. Briefly, rats were sacrificed by CO₂ asphyxiation and bathed in 70% ethanol, and two 4- × 4-cm pieces of skin were removed from the dorsal surface and placed in HBSS with 2% penicillin/streptomycin. Skin was cultured immediately.

Subculture method. Briefly, medium was removed from the dishes and the cells were washed three times with 5 ml of fresh HBSS. Cells were detached with 3 ml of 0.25% trypsin (Gibco) and incubated at 37°C for 4 to 6 min. The trypsin action was then stopped by addition of 3 ml of FBS. The liquid was transferred to sterile polyethylene test tubes and centrifuged at 1000 × g at 4°C for 10 min. The supernatant was removed, and cells were resuspended in 5 ml of medium and transferred to 150-cm² culture flasks containing 20 ml of medium. Flasks were incubated for 1 to 2 weeks until cultures became confluent.

Preparation of fibroblast homogenates. Briefly, to detach cells, confluent cultures (6-8/treatment group) were incubated with 12 ml of trypsin for 4 to 6 min at 37°C and then stopped with 12 ml of FBS. The cells were pelleted by centrifugation as described above, resuspended in 1 ml of PBS and hand-homogenized using a glass 5-ml tissue grinder (Mandel Scientific Ltd., Guelph, Ontario, Canada). Homogenates were separated into 100-µl aliquots, frozen in liquid nitrogen and stored at 80°C until DNA was isolated.

Micronucleus Formation

The cells were incubated with either phenytoin at 80 µM or HPPH at 10 or 80 µM for 5 hr, at which point the cells were washed three times with 5 ml of HBSS. Fresh medium (5 ml) was then added, and cells were allowed to undergo one complete mitotic cycle (26 hr) (Vienneau et al., 1995), after which the medium was aspirated off and the cells were washed three times with 5 ml of HBSS to remove all residual medium. The 5-hr phenytoin or HPPH incubation was included as part of the mitotic cycle. To fix the cells, 5 ml of formalin solution (37% formaldehyde solution/PBS, 1:9, v/v) was added to the cells. After 30 min, the formalin solution was aspirated off and the cells were washed three times with 5 ml of PBS. Control cells were treated with the HPPH and phenytoin vehicle DMSO. Once fixed, the cells were stained with 5 ml of 4,6-diamidino-2-phenylindole fluorescent stain (2 µg/ml in water), and 2000 mononucleated cells were counted for the formation of micronuclei, using an inverted microscope with a 40× objective.

DNA Oxidation

To determine the potential role of DNA oxidation as a molecular mechanism in HPPH-initiated micronucleus formation, +/+ or +/j fibroblasts were incubated with or without HPPH (80 µM), as described above for the micronucleus studies. The cells were harvested and homogenized after one mitotic cycle, stored at 80°C, prepared and analyzed as described below.

Fibroblast DNA isolation. A modified method of Gupta (1984) was used to isolate DNA from rat skin fibroblasts. Briefly, fibroblast homogenates were incubated overnight with proteinase K at 55°C. Tris-HCl (1 mM) at a volume of 25 µl was added, and DNA was extracted with 1 volume of chloroform/isoamyl alcohol/phenol (24:1:25) and then twice with 1 volume of chloroform/isoamyl alcohol (24:1). At each stage, mixtures were vortex-mixed for 30 sec and microcentrifuged at 18,000 × g for 1 min (model E; Beckman) to separate extraction phases. The DNA was then precipitated with 500 µl of 100% ice-cold (20°C) ethanol and pelleted by microcentrifugation for 1 min. The DNA pellet was dissolved in 500 µl of phosphate buffer (pH 7.4) and incubated at 37°C on a rocker platform, with ribonuclease A (100 µg/ml) and ribonuclease T₁ (50 units/ml) to digest residual RNA. One volume of chloroform/isoamyl alcohol (24:1) was used to reextract the dissolved DNA, and the sample was microcentrifuged for 1 min. The DNA was reprecipitated as described above. The pellet was redissolved in 500 µl of 20 mM sodium acetate buffer (pH 4.8) and quantified using a UV/visible spectrophotometer (Lamda 3; Perkin Elmer Canada Ltd.) at a wavelength of 260 nm, with calf thymus DNA as the standard. The DNA was then digested to nucleotides by incubation with nuclease P₁ (67 µg/ml) at 37°C for 30 min, followed by a 60-min incubation with E. coli alkaline phosphatase (0.37 U/ml) at 37°C. The mixture of nucleosides was syringe tip-filtered (0.22 µm) and analyzed via HPLC coupled with electrochemical detection (Shigenaga and Ames, 1991).

DNA oxidation and analysis. DNA oxidation (8-OH-2-dG) was quantified using an isocratic HPLC system (Scientific Systems, Inc., State College, PA) equipped with an electrochemical detector (model 5100A, Coulochem; ESA, CA), a reverse-phase C₁₈ column (Jones Chromatography, Lakewood, CO) and a recording integrator (model CR501 Chromatopac; Shimadzu, Kyoto, Japan). Samples were eluted using a mobile phase consisting of 50 mM KH₂PO₄ buffer (pH 5.5) and 10% methanol, at a flow rate of 0.8 ml/min, with an oxidation potential of +0.4 V.

Embryo Culture

Embryo preparation. The embryo culture method has been described in detail elsewhere (Winn and Wells, 1995b). Male rat serum contains undefined nutrients and factors required by murine embryos for survival and growth; therefore, it was used as the medium in which the embryos were cultured. Blood was obtained, as described elsewhere (Winn and Wells, 1995b), from retired CD-1 male rat breeders (Charles River). The blood was centrifuged for 5 min at 1000 × g at 4°C (model TJ-6; Beckman Instruments, Toronto, Ontario, Canada) and kept on ice until blood was obtained from all animals. All blood samples were then centrifuged for 30 min at 1900 × g at 4°C (model J2-21M; Beckman Instruments). To evaporate residual protein-bound ether, pooled serum was heat-inactivated for 1 hr at 58°C and gassed (5% CO₂ in air; Cannox Canada, Toronto, Ontario, Canada) for 30 min. The heat-inactivated male rat serum was divided into aliquots and stored at 80°C.

Developmental parameters. Developmental parameters included dorsal-ventral flexure (turning), anterior neuropore closure and somite development. Because somite development can be correlated with discrete and distinct developmental events and is directly related to the growth and development of the embryo, somite development in each embryo was assessed. Somite development of individual embryos was determined by subtracting the number of somites present at the termination of the culture from the somite count noted at the beginning of each culture. The final somite count was determined by counting from the location of the anterior limb bud (13th somite) in a cranial-to-caudal direction. This technique was used because somites cranial to the 13th somite begin to disperse in preparation for future morphological development, making accurate somite determination difficult.

Morphological parameters. Morphological assessment included determination of yolk sac diameter (in millimeters) and crown-rump length (in millimeters). The measurement of yolk sac diameter was made at the widest point perpendicular to the ectoplacental cone. Measurements were made at either 3.2× or 4.0× magnification, with an eyepiece reticle micrometer. The crown-rump length was defined as the distance from the mesencephalon to the lumbar-sacral region in embryos that had turned and was not measured in embryos that had not turned.

MS

Urine sample preparation. UGT-normal Wistar and +/j and j/j UGT-deficient Gunn rats were treated with a teratogenic dose of phenytoin (150 mg/kg i.p.) and housed separately in metabolic cages (Nalgene; Sybron Corp., Rochester, NY), and urine was collected over a 4-hr period. The urine samples were diluted with 10 volumes of methanol (precooled to 20°C) and were kept at 20°C for 20 min to precipitate all protein in the urine. The samples were then centrifuged (model TJ-6; Beckman Instruments) at 1000 × g for 20 min at 4°C. A 1-ml aliquot of the supernatant was passed through a 0.22-µm syringe tip filter (Millex-GS; Millipore Corp., Bedford, MA) and reduced to 50 µl under a stream of nitrogen gas, and 20 µl was then injected into an HPLC system in line with a tandem mass spectrometer.

Sample analysis. HPLC-MS (API III; Perkin-Elmer Sciex, Concord, Ontario, Canada) was used in the ion spray mode. An isocratic pump equipped with a 15-cm ODS IIC-18 column (particle size of 5 µm; Jones Chromatography) was used with a mobile phase composition of 40% acetonitrile, 59% deionized water and 1% acetic acid, at a flow rate of 1 ml/min. The collision activation spectra of the phenytoin and HPPH glucuronides were obtained using HPLC-MS/MS, with argon as the target gas, at an energy of 80 eV. The mean mass ± S.E. was calculated from the multiply charged ions by the software Mass spec. (version 3.3).

Statistical Analysis

Statistical significance of differences between treatment groups was determined by Student's t test or one-factor analysis of variance as appropriate, using a standard, computerized, statistical program (Statsview; Abacus Concepts, Inc.). The level of significance was P < .05.

	Results

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Cell Culture Studies

Concentration- and UGT phenotype-dependent increases in HPPH-initiated micronucleus formation. HPPH-initiated micronucleus formation exhibited a concentration-dependent response in all cell phenotypes, although the UGT-deficient phenotypes were substantially more susceptible (fig. 2). In +/+ UGT-normal cells enhanced micronucleus formation required 80 µM HPPH (P = .03), whereas with both +/j and j/j UGT-deficient cells nearly maximal micronucleus formation was initiated with only 10 µM HPPH. The magnitude of micronucleus formation initiated by 10 µM HPPH was equivalent in +/j and j/j UGT-deficient cells. These +/j and j/j UGT-deficient cells treated with 10 µM HPPH showed 4.0-fold and 3.8-fold increases, respectively, in micronucleus formation, compared with respective DMSO-treated phenotypes (P = .0001), and 2.7-fold and 3.0-fold enhancements, respectively, compared with the increase observed in comparable HPPH-treated +/+ UGT-normal cells (P = .007, P = .0002). Compared with respective DMSO-treated phenotypes, micronucleus formation initiated by 80 µM HPPH was increased 2.0-fold, 4.8-fold and 4.6-fold in +/+ UGT-normal (P = .03) and +/j and j/j UGT-deficient cells, respectively (P = .0001) (fig. 2). There also were 3.0-fold and 3.4-fold enhancements in micronucleus formation initiated by 80 µM HPPH in +/j (P = .02) and j/j (P = .04) UGT-deficient cells, respectively, compared with the increase in HPPH-treated +/+ UGT-normal cells (fig. 2). In DMSO-treated cells, micronucleus formation was not different among the UGT phenotypes.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of UGT deficiencies and concentration of HPPH on micronucleus formation. Skin fibroblasts were cultured from either homozygous +/+ UGT-normal or heterozygous +/j or homozygous j/j UGT-deficient Gunn rats. Cells were incubated with HPPH (10 or 80 µM) for 5 hr, washed and cultured for the rest of one mitotic cycle (26 hr). Cells were fixed and stained, and micronuclei were counted. Symbols indicate the mean of four fibroblast cultures. *, difference from DMSO controls (P < .05). +, difference from similarly treated +/+ cells (P < .05). U, difference from 10 µM HPPH-treated +/+ cells (P < .05).

Comparative genotoxic potencies of phenytoin and HPPH. At equimolar concentrations (80 µM), phenytoin and HPPH initiated similar increases in micronucleus formation (fig. 3). Compared with respective DMSO-treated controls, micronucleus formation initiated by 80 µM phenytoin was increased 1.7-fold, 4.4-fold and 3.8-fold in +/+ UGT-normal (P = .03) and +/j and j/j UGT-deficient (P = .0001) cells, respectively. There was a >3.9-fold increase in phenytoin-initiated micronucleus formation in both +/j (P = .006) and j/j (P = .009) UGT-deficient cells, compared with the increase observed in phenytoin-treated +/+ UGT-normal cells.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Comparison of the potencies of phenytoin and HPPH in initiating micronucleus formation. Skin fibroblasts were cultured from either homozygous +/+ UGT-normal or heterozygous +/j or homozygous j/j UGT-deficient Gunn rats. Cells were incubated with 80 µM phenytoin or HPPH for 5 hr, washed and cultured for the rest of one mitotic cycle (26 hr). Cells were fixed and stained, and micronuclei were counted. Bars indicate the mean of four fibroblast cultures. *, difference from DMSO controls (P < .05). +, difference from +/+ UGT-normal cells (P < .05).

HPPH-initiated DNA oxidation. 8-OH-2-dG was increased in j/j UGT-deficient cells treated with 80 µM HPPH, compared with both HPPH-treated and DMSO-treated +/+ UGT-normal cells (fig. 4) (P < .05).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of UGT deficiency on DNA oxidation initiated by HPPH. DNA oxidation was quantified by the formation of 8-OH-2-dG. Skin fibroblasts were cultured from either homozygous +/+ UGT-normal or homozygous j/j UGT-deficient Gunn rats. Cells were incubated with 80 µM HPPH for 5 hr, washed, harvested and analyzed for 8-OH-2-dG. Bars indicate the mean of four cultures. *, difference from HPPH-treated +/+ cells (P < .05).

Embryo Culture Studies

Similarly to results from cell culture/micronucleus studies, mouse embryos exposed for 24 hr to 10 µM HPPH did not demonstrate embryotoxicity, compared with vehicle controls (fig. 5). However, upon incubation with 80 µM HPPH, there was significant dysmorphogenesis, as evidenced by decreases in anterior neuropore closure (45%), turning (35%), yolk sac diameter (8%) and crown-rump length (9%) (P < .05) (fig. 5). These embryopathic effects of 80 µM HPPH were equivalent to those reported with phenytoin at an identical concentration (Winn and Wells, 1995b), which also is within the therapeutic range of phenytoin in maternal plasma (Winn and Wells, 1995a). Interestingly, unlike phenytoin (Winn and Wells, 1995b), HPPH did not significantly reduce somite development.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Embryotoxicity of HPPH in CD-1 mouse embryo culture. Embryos were cultured on gestational day 9.5 in the presence of either HPPH [2.7 µg/ml (10 µM) or 21.5 µg/ml (80 µM)] or the vehicle (0.002 N NaOH) for 24 hr. The concentration of 80 µM is equimolar both to that for phenytoin known to be embryopathic in mouse embryo culture (Winn and Wells, 1995b) and to the therapeutic concentration of phenytoin in maternal plasma. The number of embryos is given in parentheses. *, difference from vehicle controls (P < .05).

HPLC-MS/MS

Analysis of urine samples from UGT-normal Wistar and +/j UGT-deficient Gunn rats by HPLC-MS/MS showed a parent ion at m/z 429, with a retention time of 1.87 min. This compound was designated as the N³-glucuronide of phenytoin (fig. 1), based on a number of experimental observations. MS analysis of this parent ion resulted in the fragmentation pattern shown in figure 6. An N³-glucuronide conjugate of phenytoin in bile extract from Wistar rats was previously reported (Smith et al., 1977). In that study, ions that appeared at m/z 322 and 378 were said to arise from retro-Diels-Alder rearrangements of the glycone ring. Our studies are consistent with the previously reported fragmentation patterns of an N³-glucuronide of phenytoin, with the exception of an m/z value of 337 (m/z 378 in the study by Smith et al.). This discrepancy likely was due to a different method of ionization (ion spray) used in our mass spectrometer. Importantly, the N³-glucuronide of phenytoin was not detected in the urine of j/j UGT-deficient Gunn rats.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Collision activation spectrum of the N3-glucuronide of phenytoin observed in the urine of Wistar and heterozygous +/j Gunn rats, including the structures assigned to each fragment. Ph, phenyl.

In the urine of Wistar rats, as expected, a parent ion with a retention time of 1.42 min and an m/z value of 445 was evident, corresponding to the O-glucuronide of HPPH, the major, para-hydroxylated metabolite of phenytoin (fig. 1). Importantly, this parent ion reflecting O-glucuronidation of HPPH was not observed in the urine of either +/j or j/j UGT-deficient Gunn rats.

	Discussion

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Phenytoin is therapeutically and toxicologically important due to its antiepileptic efficacy and teratogenic potential, respectively. Although phenytoin is teratogenic in many animal species, including humans (Winn and Wells, 1995a), the danger to both the mother and fetus from uncontrolled seizures is considered to be greater than the possible teratogenic effects of phenytoin, and therapy generally is continued throughout pregnancy. UGTs are known to catalyze the glucuronidation and elimination of both phenytoin (Smith et al., 1977) and its major, para-hydroxylated metabolite, HPPH. This study demonstrated that phenytoin and HPPH were equipotent initiators of micronucleus formation in rat skin fibroblasts. This genotoxic outcome may reflect teratological initiation, as has been postulated for carcinogenic initiation, because HPPH also proved in mouse embryo culture to be equipotent to phenytoin in embryotoxicity. Furthermore, UGT deficiencies resulted in decreased glucuronidation of phenytoin and HPPH, with a resultant enhancement in phenytoin- and HPPH-initiated DNA oxidation and micronucleus formation, suggesting that hereditary UGT deficiencies may play an important role in teratological susceptibility.

There have been several hypotheses proposed for the mechanism of phenytoin-initiated teratogenesis (Hansen, 1991; Juchau et al., 1992; Winn and Wells, 1995a), including PHS- and/or lipoxygenase-catalyzed bioactivation to a reactive intermediate (Winn and Wells, 1995a). PHS and lipoxygenases produce prostaglandins, leukotrienes and related eicosanoids from polyunsaturated fatty acids such as arachidonic acid. In this synthetic pathway, xenobiotics such as phenytoin (Smith et al., 1991; Winn and Wells, 1995a) and HPPH (C. J. Nicol and P. G. Wells, unpublished results) can donate an electron and thus be oxidized to a free radical intermediate (Winn and Wells, 1995a; Parman et al., 1996) (fig. 1).

This study in rat skin fibroblasts showed that HPPH could initiate DNA oxidation and genotoxicity, the latter reflected by enhanced micronucleus formation, and that these effects were enhanced >3-fold in UGT-deficient cells. Thus, as has been postulated for phenytoin itself (Winn and Wells, 1995a,b), HPPH may contribute to the teratogenicity of phenytoin via the same mechanism of peroxidase-catalyzed bioactivation and reactive oxygen species-mediated oxidative damage to DNA and other targets (fig. 1). Similar results were seen in an in vitro horseradish peroxidase-H₂O₂ system, where both phenytoin and HPPH initiated the formation of 8-OH-2-dG (Winn and Wells, 1995a). The results of the present study suggest that phenytoin-initiated DNA oxidation (Winn and Wells, 1995a) and hydroxyl radical formation (Kim and Wells, 1996b) may be mediated in part by HPPH bioactivation to a reactive free radical intermediate, which, similarly to B[a]P (Kim and Wells, 1995, 1996a), may constitute a molecular mechanism for both phenytoin- and HPPH-initiated micronucleus formation and, potentially, teratogenesis.

The studies in mouse embryo culture constitute the first direct evidence for an embryotoxic effect of HPPH, contrary to results from previous in vivo studies discussed below. HPPH was equipotent to phenytoin (Winn and Wells, 1995b) in this regard, initiating embryopathic effects at a concentration of 80 µM, which is within the therapeutic concentration range for phenytoin in maternal plasma. The spectrum of embryopathic effects for HPPH also was almost identical to that for phenytoin in embryo culture (Winn and Wells, 1995b), including decreases in anterior neuropore closure, turning, yolk sac diameter and crown-rump length. The only exception was no decrease in somite development, which for phenytoin is small but usually statistically significant (Winn and Wells, 1995b). At a lower HPPH concentration (10 µM) more likely to be encountered in vivo, where up to 93% (Chow and Fischer, 1982) of HPPH can be glucuronidated, HPPH exhibited no significant embryotoxicity in our embryo culture model, although the possibility of other embryopathic effects, such as neurotoxicity, from in vivo exposure to such lower concentrations of HPPH cannot be excluded. An embryopathic contribution from HPPH would be more likely in UGT-deficient mothers, in whom decreased glucuronidation of HPPH would lead to greater embryonic exposure to this potentially embryotoxic metabolite, as discussed below (fig. 1).

The potential teratological contribution of HPPH during phenytoin therapy is particularly remarkable because equivalent genotoxicity was observed with only 10 µM HPPH, which is about one-tenth of the maternal therapeutic concentration for phenytoin (80 µM). The lower concentration of HPPH (10 µM) was genotoxic only in UGT-deficient cells and not in UGT-normal cells. UGTs were substantially genoprotective, suggesting that the reported apparent lack of in vivo HPPH teratogenicity (Harbison and Becker, 1974) and genotoxicity (Barcellona et al., 1987) was due to maternal glucuronidation, preventing HPPH from reaching the embryo. If so, then pregnant women with certain hereditary UGT deficiencies may be at increased risk for the teratogenicity of phenytoin and related xenobiotics that are eliminated substantially via glucuronidation. Evidence from pregnant UGT-deficient Gunn rats, which show enhanced susceptibility to B[a]P embryotoxicity (Wells et al., 1989), suggests that UGT deficiencies are teratologically relevant. Human studies with acetaminophen in vivo (de Morais et al., 1992b) and with B[a]P in an in vitro human lymphocyte model (Hu and Wells, 1993) indicate that human UGT deficiencies are relatively common and result in decreased xenobiotic glucuronidation with enhanced bioactivation and cytotoxicity.

Although this study presents the first evidence for HPPH-initiated DNA oxidation, micronucleus formation and embryotoxicity, phenytoin itself has been shown to initiate DNA oxidation in embryo culture (Winn and Wells, 1995b) during a gestational time when embryos have little or no demonstrable P450 for forming HPPH in situ. This study does present the first evidence for phenytoin-initiated micronucleus formation, which is consistent with its ability to irreversibly damage DNA via both oxidation and arylation (Winn and Wells, 1995a,b). The teratological relevance of DNA damage by phenytoin and HPPH is further supported by the enhanced teratogenicity of phenytoin in p53-deficient mice, which have compromised DNA repair (Laposa et al., 1996). A similarly enhanced teratological susceptibility of p53-deficient mice was observed for B[a]P, another DNA-damaging teratogen and carcinogen (Nicol et al., 1995). An equivalent enhancement in micronucleus formation and embryotoxicity initiated by phenytoin and HPPH was not surprising, and a similar equivalence was reported using a rat embryo limb culture assay (Brown et al., 1986). However, the enhanced genotoxicity of phenytoin itself in UGT-deficient cells was unexpected, because we were not aware that phenytoin could be directly glucuronidated. Although phenytoin potentially could be hydroxylated by P450s to HPPH, for which UGTs are expected to be protective, P450 activities in rat skin fibroblasts are negligible (Vienneau et al., 1995; Kim and Wells, 1995), and this is an unlikely explanation for UGT protection against the observed in vitro genotoxicity of phenytoin. Also, phenytoin itself has been shown to oxidize DNA in embryo culture (Winn and Wells, 1995b), as discussed above. Thus, the only apparent mechanism for UGT-dependent protection against the genotoxicity of phenytoin itself is via direct glucuronidation of phenytoin. This hypothesis was evaluated by HPLC-MS/MS analysis of the urine from UGT-normal Wistar and UGT-deficient Gunn rats treated with a teratogenic dose of phenytoin. An N³-glucuronide conjugate of phenytoin was identified in UGT-normal rats; we subsequently discovered it had been reported previously in Wistar rats by Smith et al. (1977). More importantly, we found that the N³-glucuronide of phenytoin was not detected in j/j UGT-deficient Gunn rats and the O-glucuronide of HPPH was not detected in either +/j or j/j UGT-deficient Gunn rats. These results provide the first evidence that UGT deficiencies lead to reduced in vivo glucuronidation of both phenytoin and its HPPH metabolite. Assuming a similar process in fibroblasts, as has been shown for B[a]P (Vienneau et al., 1995), these results suggest that decreased glucuronidation resulted in enhanced DNA oxidation and genotoxicity initiated by both phenytoin and HPPH in UGT-deficient fibroblasts.

For both phenytoin and HPPH, maximal genotoxic susceptibility was observed in +/j UGT-deficient cells, with no further enhancement in j/j UGT-deficient cells. This suggests that these concentrations in UGT-deficient cells constitute the plateau of the concentration-response curve. Similar results were seen both in vitro (Vienneau et al., 1995; Kim and Wells, 1996a) and in vivo (de Morais et al., 1992a; Hu and Wells, 1992, 1994), where +/j UGT deficiencies increased acetaminophen bioactivation and toxicity, as well as B[a]P- and NNK-initiated micronucleus formation. These results suggest that hereditary UGT deficiencies may have considerable clinical relevance, because, unlike homozygous deficiencies, heterozygous deficiencies are relatively common.

In bacterial studies, mutagenicity initiated by both phenytoin and HPPH was shown to be dependent upon P450-catalyzed enzymatic bioactivation, requiring preincubation with a metabolic activating system (S9 liver fraction) (Sezzano et al., 1982). Phenytoin was mildly mutagenic in the TA 1538 strain of Salmonella typhimurium at 25 µg (38 µM) and 250 µg (381 µM)/2.6 ml/plate upon preincubation with S9 from rats induced with the P450 inducers 3-methylcholanthrene and aroclor 1254, respectively. HPPH was more mutagenic than phenytoin after preincubation with similar S9 fractions, including S9 fractions from rats induced with -naphthoflavone at HPPH concentrations ranging from 25 (36 µM) to 250 (358 µM) µg/2.6 ml/plate. However, a conflicting study conducted under similar conditions found that phenytoin (25-1000 µg/2.6 ml/plate, 38-1524 µM) and HPPH (25-500 µg/2.6 ml/plate, 36-717 µM) were not mutagenic in all strains (TA97, TA98, TA100, TA1530, TA1537 and TA1538) tested (Leonard et al., 1984). Similar contradictory results were reported for in vivo sister chromatid exchange in phenytoin-treated patients. Hadebank et al. (1982) found a significant increase in sister chromatid exchange in patients undergoing phenytoin monotherapy, whereas Hunke and Carpenter (1978) did not see a difference in patients with phenytoin serum concentrations ranging from 3.8 µg/ml (15 µM) to 29.5 µg/ml (117 µM). However, in vitro studies by Hunke and Carpenter (1978) found that phenytoin concentrations ranging from 10 µg/ml (40 µM) to 100 µg/ml (396 µM) significantly increased sister chromatid exchange, suggesting that phenytoin and/or its metabolite HPPH is mutagenic and genotoxic.

In our study, phenytoin and HPPH at the equivalent of a human therapeutic concentration for phenytoin (80 µM) were equipotent in initiating micronuclei in skin fibroblasts cultured from +/+ UGT-normal Gunn rats vs. DMSO-treated controls. In vivo, phenytoin at doses of 0.5 and 1.0 mg/kg, but not 6 to 20 mg/kg, initiated micronuclei in mouse bone marrow polychromatic erythrocytes (Montes de Oca-Luna et al., 1984). A somewhat contradictory in vivo study found that 100 mg/kg phenytoin initiated micronucleus formation only in fetal (day 13), and not in maternal, polychromatic erythrocytes (Barcellona et al., 1987). Furthermore, a molar equivalent dose (106 mg/kg) of HPPH did not initiate micronuclei in either fetal or maternal erythrocytes (Barcellona et al., 1987), substantiating an earlier study showing that HPPH administered in vivo at the molar equivalent of a teratogenic dose of phenytoin did not initiate teratogenesis in mice (Harbison and Becker, 1974). In contrast, our study found not only that HPPH could initiate micronucleus formation in +/+ UGT-normal rat skin fibroblasts but also that micronucleus formation was increased in +/j and j/j UGT-deficient fibroblasts treated with either 80 µM phenytoin or HPPH (figs. 2 and 3). During phenytoin therapy, approximately 60% of HPPH normally would be glucuronidated (Browne and Chang, 1989), and in mice >90% is glucuronidated (Chow and Fischer, 1982); however, even 10 µM HPPH was as genotoxic as the 80 µM concentration in UGT-deficient cells and thus may contribute to genotoxicity, particularly in UGT-deficient people.

In summary, these results suggest that DNA oxidation may constitute a molecular mechanism for the initiation of micronuclei by both HPPH and phenytoin, as has been postulated for phenytoin teratogenicity (Winn and Wells, 1995a,b), and also may constitute a mechanism for HPPH embryotoxicity and other adverse effects. The results in mouse embryo culture provide the first direct evidence for HPPH-initiated embryotoxicity, the potency of which was equivalent to that previously reported for phenytoin (Winn and Wells, 1995b). UGTs provided important protection against both phenytoin- and HPPH-initiated in vitro genotoxicity, and related in vivo studies with B[a]P (Wells et al., 1989) suggest that these in vitro results have teratological relevance. The genotoxicity of both phenytoin and HPPH was as high in heterozygous +/j as in homozygous j/j UGT-deficient cells, suggesting that hereditary UGT deficiencies may have considerable relevance to clinical toxicological susceptibility. However, although UGT-deficient animal models can reflect some human UGT deficiencies and their toxicological consequences, further studies will be necessary to confirm the relevance of these results to teratological susceptibility, particularly in humans.