Effects of Phenytoin on Glutathione Status and Oxidative Stress Biomarker Gene mRNA Levels in Cultured Precision Human Liver Slices

Evan P. Gallagher1 and Karen M. Sheehy

Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, P.O. Box 110885, Gainesville, Florida 32611-0885

Received April 6, 2000; accepted September 26, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular production of reactive oxygen species (ROS) has been implicated as an important mechanism of chemical teratogenesis and developmental toxicity. Unfortunately, the lack of relevant model systems has precluded studies targeting the role of ROS in human teratogenesis and prenatal toxicity. In the current study, we have used cultured precision human prenatal liver slices to study the effects of the human teratogen phenytoin (diphenylhydantoin; Dilantin) on cell toxicity, glutathione redox status, and steady-state mRNA expression of a panel of oxidative stress-related biomarker genes. The biomarker genes analyzed were p53, bcl-2, alpha class glutathione S-transferases isozymes A1 and A4 (hGSTA1 and hGSTA4), and the catalytic subunit of {gamma}-glutamylcysteine synthetase ({gamma}GCS-HS). Liver slices (200 µm) were prepared from second trimester prenatal livers and cultured in the presence of 0, 250 µM, and 1000 µM phenytoin for 18 h. Exposure to 1000 µM phenytoin elicited 41% and 34% reductions in slice intracellular potassium and reduced glutathione (GSH) concentrations, respectively. The reduction in slice GSH concentrations at 1000 µM phenytoin was accompanied by a 2.2-fold increase in the percentage of total slice glutathione consisting of GSSG, and a 3.9-fold increase in hGSTA1 steady-state mRNA expression. Exposure to 250 µM or 1000 µM phenytoin also elicited a relatively minor (less than 2-fold) but significant increase in p53 steady-state mRNA expression. In contrast, the steady-state levels of {gamma}GCS-HS, hGSTA4, and bcl-2 mRNAs were not affected by phenytoin exposure. Our findings in a relevant human model system are supportive of a protective role of GSH and hGSTA1 against phenytoin toxicity and teratogenesis. These studies also demonstrate the utility of using cultured human prenatal liver slices as a relevant tool for developmental toxicology studies.

Key Words: precision liver slices; phenytoin; mRNA expression; glutathione transferases; oxidative stress.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is estimated that epilepsy affects 680,000 to 1 million women of childbearing age in the United States (Yerby, 1994). Associated with the physiological ramifications of untreated seizures is an increased risk of birth defects in the offspring of epileptic mothers. The incidence of malformations observed in the infants of epileptic mothers can be as high as 18.6%, as compared to only 2–3% in the general population (Yerby, 1994). Phenytoin is an efficacious anticonvulsant drug that is commonly used in the treatment of grand mal seizures associated with epilepsy. Although phenytoin is teratogenic in animals and one of the few known human teratogens, its use is generally continued throughout pregnancy because of the high risk associated with untreated epileptic seizures (Cleland, 1991Go). The prenatal hydantoin syndrome describes the myriad of birth defects in infants exposed in utero to phenytoin, including mental retardation, forelimb and craniofacial malformations, and developmental defects (Wells and Winn, 1996Go). Unfortunately, approximately one-third of all infants whose mothers received phenytoin therapy will develop at least some of the defects associated with prenatal hydantoin syndrome (Cleland, 1991Go).

The mechanisms of phenytoin teratogenicity have been investigated in a number of in vitro and in vivo animal models (Finnell et al., 1999Go; Kindig et al., 1992Go; Ozolins et al., 1996Go; Winn and Wells, 1995Go, 1999Go; Yu and Wells, 1995Go) and also in embryo culture (Winn and Wells, 1995Go). For example, antioxidants such as superoxide dismutase (SOD), catalase, and GSH confer protection against phenytoin embryopathy in vitro (Winn and Wells, 1995Go) and in vivo (Winn and Wells, 1999Go). Dietary deprivation of selenium in mice results in a loss of selenium-dependent GSH peroxidase activity in maternal and embryonic tissues and an increased susceptibility to phenytoin teratogenicity (Winn and Wells, 1997Go). Culture of murine embryos with a clinically relevant dose of phenytoin (80 µM) causes oxidative DNA damage as evidenced by the formation of 8-hydroxy-2'-deoxyguanosine (Winn and Wells, 1995Go). Collectively, these studies implicate reactive oxygen species (ROS) and their products as causative agents in phenytoin teratogenesis. In addition, the teratological contribution of other mechanisms, such as covalent binding of phenytoin intermediates, is hypothesized to be involved in phenytoin teratogenesis (Wells and Winn, 1996Go). Consequently, it is likely that more than one molecular mechanism is involved in phenytoin teratogenesis, and that the mechanisms may vary with the cell type, tissue, and gestational time of exposure and species.

Despite the wealth of information generated from animal studies, the lack of relevant human models adds uncertainty to the understanding of the mechanisms of phenytoin teratogenesis to humans. In this regard, human liver slices prepared from adult donors have been used in a number of toxicological applications (Beamand et al., 1996Go, 1997Go; Lake et al., 1996aGo,bGo) and provide an alternative in situ technique for studying drug metabolism. Recently, investigators have extended the use of the human liver slice culture model to examine the effects of chemicals on induction of biotransformation enzymes (Drahushuk et al., 1996Go; Lake et al., 1996bGo). The current study was initiated to determine the feasibility of using cultured prenatal liver slices as a tool to investigate the mechanisms of prenatal phenytoin toxicity in humans. Because the prenatal liver differs markedly with respect to physiology and drug metabolism enzyme expression when compared to fully mature adult liver, our goal was to apply a liver slice culture system to studies applicable to the in utero environment. We selected phenytoin as a model drug teratogen for our prenatal liver slice study based upon the importance of the liver as a target organ for phenytoin biotransformation and toxicity (Gabler and Hubbard, 1972Go; Hansen and Hodes, 1983Go; Rane and Peng, 1985Go) and because of the role of oxidative stress as a relevant mechanism of phenytoin teratogenesis. In the current study, we examined the effects of phenytoin exposure on tissue slice viability, GSH/GSSG redox status, and steady-state mRNA levels of candidate biomarker genes involved in cellular oxidative stress. Included in our panel of biomarker genes were hGSTA1 and hGSTA4, and {gamma}GCS-HS, which encodes the catalytic subunit of the first and rate-limiting enzyme in GSH biosynthesis (Gipp et al., 1992Go). Because of the mechanistic links among oxidative stress, apoptosis, and DNA damage, we analyzed for potential effects on steady-state mRNAs encoding bcl-2 (Morales et al., 1998Go) and the tumor suppressor gene p53 (Duerksen-Hughes et al., 1999Go). The results of this study demonstrate the utility of using cultured prenatal liver slices as a toxicological tool to study drugs and chemicals of relevance to human transplacental exposure and are supportive of a role of hGSTA1 and GSH in protecting against phenytoin-mediated cell injury in humans.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Tissue culture media, media components, PCR components, agarose, Trizol, nylon membranes, TAQ polymerase, and other molecular biology reagents were purchased from Gibco-BRL (Gaithersburg, MD, USA). Phenytoin (5,5-diphenylhydantoin), DMSO, NADP+, GSH, GSSG, and other buffers, enzymes, and cofactors were purchased from Sigma Chemical Co. (St. Louis, MO). [{alpha}-32P]dCTP was purchased from NEN (Boston, MA, USA).

Tissue samples.
All human tissue work was approved by the University of Florida Health Center Institutional Review Board. Second trimester prenatal liver samples (age 19–23 weeks) were obtained from the Anatomical Gift Foundation (AGF), an independent nonprofit human tissue bank that provides human tissue for biomedical research. All prenatal livers were obtained through elective termination of pregnancy. Strict confidentiality was maintained with respect to donor information. Because the specimens obtained by the tissue banks would otherwise be discarded, and because they are from anonymous donors, these studies are exempted by regulatory provisions regarding human subject research (45 CFR 46). The livers were immediately placed in ice-cold V-7 buffer (Fisher et. al., 1995Go) and shipped on wet ice to our laboratory at the University of Florida. Liver slices were prepared within 30 h of excision.

Preparation and culture of liver slices.
Tissue cores from liver samples were prepared using an 8-mm diameter tissue coring tool. The cores were then loaded into a Krumdieck tissue slicer (Alabama Research and Development Corp., Munford, AL, USA) containing ice-cold V-7 buffer. Precision-cut 200-µm liver slices were prepared using an oscillating blade. The slices were floated onto the stainless steel mesh screen of titanium roller inserts and placed in 20-ml glass scintillation vials containing 1.7 ml oxygenated culture medium. The culture medium consisted of Waymouth's media containing 10% fetal calf serum, 50 µg/ml gentamicin, 2.5 µg/ml fungizone, and 50 µg/ml penicillin-streptomycin. The vials were then closed with ventilated caps and placed in a dynamic organ culture incubator (Vitron, Inc., Tucson, AZ, USA) at 37°C under 95% O2/5% CO2. Slices were preincubated for 3 h in all experiments to allow for normalization to culture conditions. In preliminary viability experiments, inserts were transferred to vials containing fresh medium. Liver slices were removed at 0, 18, and 24 h, weighed, snap-frozen in liquid nitrogen, and stored at –80°C until analysis. In the phenytoin experiments, following preincubation, the inserts were transferred to vials containing phenytoin (250 µM or 1000 µM in DMSO) or DMSO alone (1% final volume) in culture medium. After an additional 18 h of incubation, the slices were weighed, snap-frozen in liquid nitrogen, and stored at –80°C for viability and Northern blotting analyses.

Analysis of GSH/GSSG and K+.
Slices used for determination of GSH/GSSG and K+ concentrations were sonicated in 9 volumes of 5% sulfosalicylic acid and centrifuged at 10,000 rpm for 10 min. Supernatants were directly analyzed for total glutathione (GSH and GSSG) and for GSSG using an enzymatic recycling assay adapted for a 96-well microplate reader (Baker et al., 1990Go). Slice K+ concentrations were determined using flame photometry on a 1:5 dilution of the acidified supernatants as described (Fisher et al., 1995Go). All assays were performed in triplicate on individual liver slices.

Polymerase chain reaction (PCR) and cDNA isolations.
cDNAs against human bcl-2, p53, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs were generously provided by Dr. Sandy Kirchner from the University of Washington. Primers used to isolate cDNA for hGSTA1 and hGSTA4 were based upon the published sequences of the open reading frame of the hGSTA1 cDNA (Genbank accession number S49975; Stenberg et al., 1992Go) and hGSTA4 cDNA (Genbank accession number Y13047; Hubatsch et al., 1998Go). Because the cDNAs encoding the hGSTA1 and hGSTA2 subunit proteins differ by only 11 amino acids, it is not possible to distinguish between hGSTA1 and hGSTA2 transcripts using cDNA probes. Approximately 90% of the sum total alpha class hGSTA1 and hGSTA2 subunit expression in human prenatal livers age 10 to 42 weeks can be accounted for by hGSTA1 (Strange et al., 1989Go). Accordingly, we will subsequently refer to mRNAs recognized by our hGSTA1 cDNA probe in our Northern blotting experiments as hGSTA1. Because the cDNA encoding hGSTA4 is considerably diverged from the other human alpha class GSTs, it was possible to construct a cDNA probe that specifically recognizes hGSTA4 mRNA in Northern blotting experiments. Primers used to isolate a human {gamma}GCS-HS cDNA were based upon the published sequences of the open reading frame of the {gamma}GCS-HS cDNA (Genbank accession number M90656; Gipp et al., 1992Go). The following primer sequences were used: hGSTA1 PCR primers: 5'-GAT-CCT-CCT-TCT-GCC-CGT-AT-3' and 5'-GGC-CTC-CAT-GAC-TGC-GTT-AT-3' for hGSTA1; 5'-ATG-GCA-GCA–AGG-CCC-AAG-CTC-CAC-TAT C-3' and 5'-TTA-TGG-CCT-AAA-GAT-GTT-GTA-GAC-GGT-TCT-3' for hGSTA4; and 5'-GAA-ACC-AAG-CGC-CAT-GC-3' and 5'-TAA-GGT-ACT-GAA-GCG-AGG-GTG-3' for {gamma}GCS-HS.

Total RNA was extracted from liver slices using Trizol reagent (Life Technologies Inc., Gaithersburg, MD) according to the manufacturer's instructions. First-strand cDNA was reverse transcribed from human liver slice total RNA using RETROscript (Ambion Inc., Austin, TX) according to the manufacturer's instructions. PCR reactions were performed in 50-µl reaction volumes containing 2 µl of total first-strand cDNA, 2.5 U Taq polymerase, 1X PCR buffer (50 mM KCl, 10 mM Tris–HCl, 2.5 mM MgCl2), 0.2 mM of each dNTP, and 0.20 µM of each 5' and 3' primer. PCR amplifications were carried out using an MJ PTC100 thermocycler. The PCR conditions included a 30-sec denaturation at 94°C followed by a 30-sec anneal at 55°C (for {gamma}GCS-HS and hGSTA1 primers) or 65°C (for hGSTA4 primers), and extension at 72°C for either 30 sec (for the {gamma}GCS-HS and hGSTA1 primers) or 60 sec (for the hGSTA4 primers). Thirty cycles were used for each gene amplification followed by a 5 min extension at 72°C. The PCR products were cloned into the pGEM-T easy plasmid vector (Promega Corp., Madison, WI) according to the manufacturer's protocol after visual confirmation of molecular weight on a 1.5% (w/v) agarose gel. The purified recombinant plasmids were sequenced by PCR cycle sequencing using the T7 (forward) and SP6 (reverse) oligonucleotide probes as sequencing primers.

Northern blotting.
Total liver RNA was electrophoresed on a 1% agarose-formaldehyde gel and transferred onto a nylon membrane. The nylon membranes were then subjected to methylene blue staining for visual inspection of 18s rRNA. The cDNA templates were labeled using Gibco's RTS RadPrime DNA Labeling System and [{alpha}-32P]dCTP. Hybridizations were performed using ExpressHyb (Clontech, Inc.) according to manufacturer's instructions. Membranes were then exposed to a phosphor screen. Quantitation of each biomarker mRNA product was carried out using ImageQuant software (Packard Instrument Company, Meriden, CT). The arbitrary density units corresponding to the tissue slice biomarker gene mRNA expression level were normalized to expression of the housekeeping gene GAPDH. The mRNAs analyzed and quantitated in the present study represent the sum of new synthesis and breakdown of RNA (e.g., steady-state mRNA expression).

Statistical analyses.
Slices were prepared from a single liver in each experiment, with incubations performed in triplicate and mean values calculated for each parameter. Values presented represent the mean ± SEM from three individual liver donors and a minimum of three separate experiments (n = 3). Significant differences in viability and mRNA expression in the control and phenytoin-treated slices were determined using ANOVA followed by the Fisher's Least Significant Differences test using Statview 4.5 (Abacus Concepts, Berkeley, CA) for Macintosh. Mean treatment-related differences were determined significant at p <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prenatal Liver Slice Viability and Redox Status
The validation of dynamic organ culture of precision cut prenatal liver slices as an in vitro model was accomplished in preliminary experiments. Approximately 5–7 viable 8-mm cores could be prepared from a typical liver tissue, yielding approximately 25–35 slices weighing approximately 20–25 mg each (data not shown). As observed in Figure 1AGo, liver slices were incubated for 24 h and remained viable throughout this period as determined by intracellular potassium retention. The potassium levels in liver slices at 18 h (75 ± 3 µmoles K+/g tissue) were 82% of initial potassium concentrations measured at time 0 (92 ± 6 µmoles K+/g tissue). Intracellular potassium retention dropped to 62% of initial levels at 24 h but were well above viable levels (> 40 µmol K+/g tissue slice; Fisher et al., 1995Go). As shown in Figure 1BGo, tissue slices maintained intracellular GSH concentrations over 24 h of culture. Tissue slice GSH concentrations increased significantly after 18 h of incubation but stabilized by 24 h (Figure 1BGo). Tissue slice GSSG concentrations after 24 h of incubation were 30 ± 3 nmol GSSG/g tissue, as compared to 4 ± 1 nmol GSSG/g tissue at time 0 (Figure 1BGo). Although tissue slice GSSG concentrations increased significantly with incubation time, the overall slice GSH/GSSG ratios remained in favor of > 95% GSH over 24 h of incubation (Figure 1BGo).



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FIG. 1. Effect of rotary culture incubation time of human prenatal liver tissue slice A) potassium concentrations, and B) tissue slice GSH and GSSG concentrations. Values are mean ± SEM of preparations from three livers. Asterisks denote values significantly different from time 0 at p <= 0.05. Dashed line in Figure 1AGo corresponds to the lower range of potassium concentrations associated with viable human liver slices (40 µmol K+/gm tissue; Fisher et al., 1995).

 
Effect of Phenytoin on Liver Slice Viability and GSH/GSSG Redox Status
A single 18-h time point was selected for analysis based upon our preliminary viability studies, published reports demonstrating phenytoin-mediated oxidative injury in animal culture under similar time frames (Winn and Wells, 1995Go, 1999Go), and limitations on the number of slices that can be prepared from relatively small prenatal liver tissues. The effect of 18-h exposure to 250 µM and 1000 µM phenytoin on the viability of liver slices is shown in Figure 2Go. Incubation of human prenatal liver slices with 250 µM phenytoin did not affect slice viability as assessed by intracellular K+ concentrations (Figure 2Go). However, exposure to 1000 µM phenytoin caused a 41% reduction in intracellular K+ concentrations. As observed in Figure 3Go, the loss in intracellular K+ by 1000 µM phenytoin was accompanied by a 34% reduction in tissue slice GSH concentrations (Figure 3AGo). A trend toward an accumulation of GSSG was observed in liver slices exposed to phenytoin, but the differences observed in slice GSSG concentrations among treatment groups were not significant at p <= 0.05 (Figure 3BGo). In contrast, the percentage of slice total glutathione accounted for byGSSG was significantly higher (2.2-fold higher) in the 1000 µM phenytoin treatment group as compared to controls (Figure 3CGo).



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FIG. 2. Effect of 18-h phenytoin exposure on the viability of cultured human prenatal liver slices as measured by tissue slice K+ concentrations. Values are mean ± SEM of preparations from three livers. Asterisks denote treatment effects that are significantly different from control slices at p <= 0.05.

 


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FIG. 3. Effect of 18-h phenytoin exposure on A) GSH concentrations, B) GSSG concentrations, and C) percentage of slice total glutathione consisting of GSSG. Values are mean ± SEM of preparations from three livers. Asterisks denote treatment effects that are significantly different than measured in control slices at p <= 0.05.

 
Northern Blotting Studies
RT-PCR of human liver total RNA using hGSTA1-specific primers produced a 374 base pair partial cDNA that was 100% homologous to bases +356 through +730 of the coding region of the previously cloned hGSTA1 cDNA (Stenberg et al., 1992Go) (sequence data not shown). In addition, the hGSTA4 PCR primers amplified a 670 base pair PCR product from human liver cDNA that was 100% homologous to bases +1 through +670 of the coding region of the hGSTA4 cDNA reported by Hubatsch et al. (1998) (sequence data not shown). Total RNA isolated from control and phenytoin-treated prenatal liver slices was hybridized with cDNA probes directed against hGSTA1, hGSTA4, {gamma}GCS-HS, p53, bcl-2, and GAPDH. Steady-state hGSTA1, hGSTA4, {gamma}GCS-HS, p53, and bcl-2 mRNA expression levels were normalized to the housekeeping gene GAPDH, whose levels were unaffected by phenytoin exposure. As observed in Figures 4 and 5GoGo, culture of liver slices in the presence of 250 µM phenytoin for 18 h caused a 1.6-fold increase in steady-state p53 mRNA expression, but did not markedly affect the steady-state mRNA expression levels of {gamma}GCS-HS, hGSTA1, bcl-2, or hGSTA4. Exposure of liver slices to a higher dose of phenytoin (1000 µM) elicited a 1.4-fold increase in p53 mRNA expression, and a 3.9-fold increase in hGSTA1 mRNA expression, but did not markedly affect {gamma}GCS-HS, hGSTA4, or bcl-2 expression (Figures 4 and 5GoGo).



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FIG. 4. Northern blot analysis of the effect of 18-h phenytoin exposure on hGSTA1, hGSTA4, {gamma}GCS-HS, bcl-2, p53, and GAPDH steady-state mRNA expression. Methylene blue stain of 18s rRNA levels are shown as a control for loading. Shown are representative Northern blots. Northern blotting was repeated twice with samples from two additional prenatal tissues in two additional experiments (n = 3 total).

 


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FIG. 5. Effect of 18-h phenytoin exposure on hGSTA1, hGSTA4, {gamma}GCS-HS, bcl-2, and p53 steady-state mRNA expression. Data represent the arbitrary density units corresponding to the tissue slice biomarker gene mRNA expression level, normalized to expression of the housekeeping gene GAPDH. Values are mean SEM of preparations from three livers and expressed as percentage of control values in the absence of phenytoin at each time point. Asterisks denote treatment effects that are significantly different than control slices at p <= 0.05. Dashed line is shown as reference for 100% of control mRNA expression in the absence of phenytoin.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The teratogenicity of many chemicals lies in part upon their bioactivation by embryonic cytochrome P450 isozymes, prostaglandin synthetases, and lipoxygenases to reactive intermediates and ROS (Autrup et al., 1984Go; Juchau, 1985Go; Juchau et al., 1986Go). These reactive metabolites can irreversibly modify critical embryonic or prenatal cellular targets, initiating a dysregulation process that can cause in utero death or teratogenesis (Wells and Winn, 1996Go). Ultimately, teratologic expression is dependent upon the embryonic intracellular balance between the processes of bioactivation and macromolecular injury, embryonic detoxification of the xenobiotic reactive intermediate(s), and repair of cellular macromolecules. The production of reactive intermediates is thought to be especially important in teratologic expression due to an unfavorable balance among these competing pathways in prenatal tissues (Wells et al., 1997Go). In the case of phenytoin, little is known about the exact mechanism of phenytoin-induced teratogenicity, and there is some debate as to the nature of the toxicologically relevant reactive intermediates produced during phenytoin metabolism (Roy and Snodgrass, 1990Go; Winn and Wells, 1997Go). For example, arene epoxides, free radical intermediates, and catechol metabolites have been implicated as causative agents in phenytoin teratogenesis. Bioactivation of phenytoin by peroxidases (Winn and Wells, 1997Go) and cytochrome P450 isozymes may represent key phenytoin bioactivation pathways. In contrast, superoxide dismutase (SOD), catalase, and GSH play a protective role in embryoprotection against phenytoin-induced protein and DNA binding (Winn and Wells, 1999Go). Maintenance of cellular GSH levels, in particular, appears to be highly important in guarding against phenytoin-mediated cell injury and teratogenesis in animals (Wong and Wells, 1989Go; Roy and Snodgrass, 1990Go; Winn and Wells, 1997Go). It appears that GSH may modulate phenytoin metabolism by either trapping a phenytoin reactive intermediate and decreasing protein binding or by protecting membrane-bound enzymes responsible for phenytoin metabolism from attack by electrophilic or free radical intermediate of phenytoin (Roy and Snodgrass, 1990Go).

In the present study, the depletion of GSH observed in prenatal liver slices incubated with 1000 µM phenytoin is supportive of a protective role of GSH against prenatal phenytoin exposure in humans. The decline of liver slice GSH on exposure to 1000 µM phenytoin was accompanied by an increase in GSSG/GSH ratios and is consistent with the aforementioned animal studies demonstrating a protective role of cellular GSH against free radical or electrophilic intermediates produced during phenytoin metabolism. Furthermore, the concomitant increase in hGSTA1 mRNA expression and decline in slice GSH concentrations in liver slices exposed to 1000 µM phenytoin suggest that GSH stores may have been partially depleted during increased hGSTA1-mediated conjugative activity. Human GST isozymes hGSTA1-1 and hGSTA4-4 are highly active toward reactive aldehydes, which are highly toxic products of peroxidative reactions (Hubatsch et al., 1998Go). We speculate that the 4-fold increase in hGSTA1 mRNA expression under conditions of high acute phenytoin exposure occurred as a protective response to remove peroxidative products resulting from oxidative injury. If so, prenatal hGSTA1-1 may represent a component of cellular defenses against phenytoin-mediated cell injury by removal of toxic products produced during phenytoin exposure. The importance of hGSTA1 in prenatal detoxification is underscored by the fact that GST isozymes containing the hGSTA1 subunit comprise approximately 90% of the total GST enzyme mass in developing human liver (Strange et al., 1989Go). In addition to its conjugative capacity, hGSTA1-1 contributes a major portion of GSH peroxidase activity in human liver and is also involved in regulating the levels of intermediates in the 5-lipoxygenase pathway (Zhao et al., 1999Go). However, whereas hGSTA1 mRNA expression has been shown to be inducible in human liver hepatocytes (Morel et al., 1993Go), little work has been published on the inducibility of hGSTA4 mRNA or hGSTA4-4 protein in human liver. In this regard, we have not observed induction of hGSTA4 mRNA expression in human adult liver slices incubated in the presence of butylated hydroxyanisole (BHA, unpublished observations), a potent GST inducer in animal models (Hayes and Pulford, 1996Go).

Inhibition of GSH biosynthesis can be another mechanism by which chemicals deplete cellular GSH. Inhibition of GSH biosynthetic capacity can occur via a decrease in the amount of GSH precursor substrates (e.g., cysteine, glycine) or by direct inhibition of {gamma}GCS mRNA expression or catalytic activity. In the present study, the decrease in tissue slice GSH on exposure to 1000 µM phenytoin was not accompanied by a loss in {gamma}GCS-HS mRNA expression at the time point analyzed. Accordingly, our data do not support inhibition of GSH biosynthesis as a mechanism underlying the loss of GSH in liver slices exposed to phenytoin. We have previously observed a reduction in {gamma}GCS-HS steady-state mRNA expression associated with reduced GCS activity and loss of GSH in rats treated with diquat and ciprofibrate (Gallagher et al., 1995Go), which are chemical agents that induce cellular oxidative stress in liver. Interestingly, previous in vivo studies (Borroz et al., 1994Go; Woods et al., 1992Go, 1999Go) and in vitro studies (Moinova and Mulcahy, 1998Go; Mulcahy et al., 1994aGo,bGo; 1997) have shown upregulation of {gamma}GCS-HS steady-state mRNA levels on chemical exposure. Thus, either negative or positive modulation of steady-state {gamma}GCS-HS mRNA expression can occur and can influence GSH homeostasis on chemical exposure. The concomitant loss of K+ and GSH in the present study likely reflects phenytoin-induced oxidative stress and a compromise of prenatal liver cellular membrane integrity, allowing for loss of small ions through cell membranes into the surrounding media. The fact that the residual tissue slice K+ concentrations in the high-dose group were well above those levels reflective of viable slices (Fisher et al., 1995Go) suggests that even the high-dose phenytoin slices remained viable throughout 18 h of exposure.

There is evidence that the tumor suppressor gene p53, which facilitates DNA repair by acting as a cell cycle checkpoint, also protects against oxidative stress-related damage in prenatal development (Wells et al., 1997Go). Induction of p53 protein, which occurs in the case of active repair, has been recently shown to be an excellent predictor of chemical-induced DNA damage (Duerksen-Hughes et al., 1999Go). In the present study, prenatal liver slice steady-state p53 mRNA expression was marginally increased after 18 h of phenytoin exposure, suggesting that a low level of phenytoin-initiated DNA damage may have been occurring at that dose. Cells that suffer severe DNA damage are eliminated through apoptosis, which can be triggered by DNA damage induced by genotoxic chemicals. Because the bcl-2 gene blocks apoptosis by inhibition of its protein product on apoptopic positive regulator proteins (Oltvai et al., 1993Go), reduced levels of bcl-2 often reflect apoptogenic action (Mirkes et al., 1997Go). The constitutive levels of bcl-2 were very low in the present study, and there were no treatmentrelated changes in bcl-2 mRNA expression. Collectively, our data suggest that maintenance of protective biochemical pathways may have afforded sufficient protection to prevent high levels of DNA damage in the slices under conditions of high-dose acute phenytoin exposure. It is also important to note that the concentrations of phenytoin used in the present study (250 µM and 1000 µM) were experimental and above those associated with relevant steady-state therapeutic levels (50–100 µM) in maternal plasma. Accordingly, our data suggest that a high single dose of phenytoin similar to those administered in our culture system may not result in human prenatal liver cell injury in vivo.

The experimental conditions used for preparation and culture of precision-cut prenatal liver slices in the present study are similar to those used by other investigators for adult human liver tissues (Fisher et al., 1995Go; Toutain et al., 1998Go). We have been able to maintain viable liver slices through 24 h of culture, which allows us an adequate time window to analyze the effects of drug exposure on viability, redox status, and gene expression. One of our concerns in establishing the prenatal liver slice culture model was to determine if extended incubation of slices in the presence of high oxygen tension (95% O2/5% CO2) would affect slice viability or cause marked intracellular oxidative damage. However, incubation of liver slices under these conditions has not appeared to cause extensive oxidative stress as demonstrated by loss of cellular GSH, changes in GSH/GSSG redox status, or loss of slice K+ concentrations. Other investigators have demonstrated that incubation of tissue slices under high oxygen tension is optimal for maintaining viability and function (Fisher et al., 1995Go; Toutain et al., 1998Go), probably due to the limitations of oxygen diffusion across the slice membranes. A significant limitation of the prenatal liver slice culture model is the relatively small number of tissue cores and viable liver slices that can be prepared from second trimester prenatal livers, as compared to human adult or rat liver preparations. Thus, the numbers of biological end points and/or time points that can be analyzed in prenatal tissue slice experiments is somewhat limited and warrant careful experimental design.

Despite the numerous advantages of using human tissue slices as an experimental model, there has been little application of tissue slice technology in developmental studies. As compared to cultured primary hepatocytes, tissue slices offer the advantage that tissue architecture is maintained, and cellular damage is relatively minimal (there is not a requirement for proteolytic enzymes in slice preparation). Unlike monolayer cell culture, tissue slices are multicell-layer aggregates in which the retention of heterogeneity and the interaction between cell types creates an environment that closely resembles the in vivo condition. The advent of commercial automated tissue slicers has allowed for the routine preparation of slices of reproducible thickness. Dynamic culture in a rotary incubator allows for control of the atmosphere to which the slices are exposed (Fisher et al., 1995Go). In particular, human tissue slices can provide mechanistic data that are highly relevant to the effects of drugs or chemicals on humans and thus reduce the uncertainty associated with human risk assessment. What is readily apparent from these studies is that dynamic organ culture of precision-cut prenatal liver slices shows promise as a relevant human model system for studying the toxic effects of transplacental drugs and chemicals. Furthermore, our system can be used to characterize effects of drug or chemical exposure on the steady-state mRNA expression of genes potentially affected by those compounds under study. From a logistical standpoint, however, it is sometimes difficult to consistently obtain high-quality tissue from commercial nonprofit tissue banks. It is therefore critical that investigators work closely with these institutions and conduct adequate validation experiments to ensure the quality and viability of tissues used in liver slice studies.

In conclusion, our studies indicate that acute exposure to high levels of phenytoin causes a loss of GSH and an increase in oxidative stress as reflected by increased GSSG/GSH ratios. Furthermore, our data suggest that induction of hGSTA1 may constitute a protective pathway by removal of oxidative damage products produced during phenytoin exposure. Collectively, these experiments demonstrate the feasibility of using cultured liver slices to determine the effects of drug exposure on tissue slice viability, redox status, and steady-state mRNA expression. Our laboratory is currently utilizing this model to investigate the effects of pro-oxidant and dietary chemicals on oxidative defense and phase II detoxification pathways in prenatal liver. Ultimately, the understanding of the ontogeny of prenatal bioactivation and protective biochemical pathways in the context of cell injury will help identify risk factors and windows of susceptibility to maternally transferred drugs and toxicants.


    ACKNOWLEDGMENTS
 
The authors gratefully knowledge Dr. Robyn Fisher of the University of Arizona for her technical assistance with the liver slice culture system and Drs. James Gardner and Adriana Doi for their technical comments on the manuscript. Funding for this project was provided by grants from the National Institutes of Health (R03-ES09380, R01-ES09427) and by the U.S. Environmental Protection Agency (R 827441).


    NOTES
 
1 To whom correspondence should be addressed. Fax: (352) 392-4707. E-mail: gallaghere{at}mail.vetmed.ufl.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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