Materials and Methods

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 providedad 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.

All following steps were conducted in a laminar flow hood. The skin was minced into 1-mm³ pieces, stored in 20 ml of HBSS (with 2% penicillin/streptomycin), transferred to sterile 100-mm polystyrene tissue culture dishes (Corning) and arranged to fit under 18-mm² coverslips. Medium (500 ml of Dulbecco’s modified Eagle medium with 75 ml of FBS and 5 ml of penicillin/streptomycin) was added at the margin of the coverslip and allowed to move across by capillary action, and then an additional 5 ml of medium was added to the dish. All dishes were then incubated at 37°C in a humidified incubator with 5% CO₂ in air and were left undisturbed for 10 days. The dishes were examined with an inverted phase-contrast microscope to confirm the formation of an epithelial layer at the margins of the skin pieces. Thereafter, 5 ml of medium was changed twice per week. After 2 months, the cultures were confluent, defined as a single layer of cells covering the bottom of the dish.

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-cm²culture 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 × gat 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.

On gestational day 9.5, pregnant murine dams were sacrificed by cervical dislocation, and embryos were explanted according to the method of New (1978). Briefly, the uterus was removed from the dam and rinsed in warmed HBSS, and the individual implantation sites were exposed using a no. 5 watchmaker’s forceps (Dumont and Fils, Montignez, Switzerland). The decidua, trophoblast, parietal endoderm and outermost membrane (Reichert’s membrane) were then removed, leaving the amnion, viseral yolk sac and ectoplacental cone intact. Explanted embryos were kept at 37°C in a holding bottle, containing pregassed (5% CO₂ in air; Cannox Canada) “holding medium” (50 ml of Waymouth’s MB 752/1 medium, 14 mM NaHCO₃, 2.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1.0 mMl-glutamine and 17 ml of male rat serum), until all embryos from all dams were explanted.

Embryos at a similar stage of development (4–6 somite pairs) were pooled and cultured in 25-cm² sterile cell culture flasks (Corning Glasswork Inc., Corning, NY), which contained 10 ml of CO₂-saturated embryo culture medium (50 ml of holding medium, 50 U/ml penicillin and 50 mg/ml streptomycin). Flasks were incubated at 37°C (model 3546 incubator, Forma Scientific, Toronto, Ontario, Canada) on a platform rocker (Bellco Biotechnology, Vineland, NJ). Embryonic morphological and developmental parameters were observed after 24 hr, using a dissecting microscope (Carl Zeiss, Oberkochen, Germany), as described below.

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.

Embryos were also examined for dorsal-ventral flexure, or turning. Gestational day 9.5 embryos are S-shaped, with the hindbody lying in the same plane as the head. After 24 hr of culture (day 10.5), under normal conditions the embryo turns, assuming a C-shaped position (fetal position) with the tail lying on the right side of the head.

To ensure proper development of the nervous system and cranial tissues, sufficient neural tube growth and neuropore closure are essential. The cranial end of the developing neural tube, from which the central nervous system develops, is called the anterior neuropore. Each embryo was examined for anterior neuropore closure, because anterior neuropore closure can be a potentially important measure of embryotoxicity, as indicated by the evidence that phenytoin can cause congenital central nervous system dysfunction in humans and animals (Winn and Wells, 1995a). Anterior neuropore closure occurs at the same time as the development of the 16th somite pair; therefore, embryos that had reached the 16th somite stage or greater without anterior neuropore closure were classified as having an open anterior neuropore.

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.