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
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ABSTRACT |
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Key Words: precision liver slices; phenytoin; mRNA expression; glutathione transferases; oxidative stress.
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INTRODUCTION |
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The mechanisms of phenytoin teratogenicity have been investigated in a number of in vitro and in vivo animal models (Finnell et al., 1999; Kindig et al., 1992
; Ozolins et al., 1996
; Winn and Wells, 1995
, 1999
; Yu and Wells, 1995
) and also in embryo culture (Winn and Wells, 1995
). For example, antioxidants such as superoxide dismutase (SOD), catalase, and GSH confer protection against phenytoin embryopathy in vitro (Winn and Wells, 1995
) and in vivo (Winn and Wells, 1999
). 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, 1997
). 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, 1995
). 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, 1996
). 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., 1996, 1997
; Lake et al., 1996a
,b
) 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., 1996
; Lake et al., 1996b
). 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, 1972
; Hansen and Hodes, 1983
; Rane and Peng, 1985
) 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
GCS-HS, which encodes the catalytic subunit of the first and rate-limiting enzyme in GSH biosynthesis (Gipp et al., 1992
). 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., 1998
) and the tumor suppressor gene p53 (Duerksen-Hughes et al., 1999
). 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.
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MATERIALS AND METHODS |
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Tissue samples.
All human tissue work was approved by the University of Florida Health Center Institutional Review Board. Second trimester prenatal liver samples (age 1923 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., 1995) 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., 1990). Slice K+ concentrations were determined using flame photometry on a 1:5 dilution of the acidified supernatants as described (Fisher et al., 1995
). 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., 1992) and hGSTA4 cDNA (Genbank accession number Y13047; Hubatsch et al., 1998
). 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., 1989
). 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
GCS-HS cDNA were based upon the published sequences of the open reading frame of the
GCS-HS cDNA (Genbank accession number M90656; Gipp et al., 1992
). 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-GCAAGG-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
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 TrisHCl, 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 GCS-HS and hGSTA1 primers) or 65°C (for hGSTA4 primers), and extension at 72°C for either 30 sec (for the
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 [-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.
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RESULTS |
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DISCUSSION |
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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., 1998). 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., 1989
). 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., 1999
). However, whereas hGSTA1 mRNA expression has been shown to be inducible in human liver hepatocytes (Morel et al., 1993
), 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, 1996
).
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 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
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
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., 1995
), which are chemical agents that induce cellular oxidative stress in liver. Interestingly, previous in vivo studies (Borroz et al., 1994
; Woods et al., 1992
, 1999
) and in vitro studies (Moinova and Mulcahy, 1998
; Mulcahy et al., 1994a
,b
; 1997) have shown upregulation of
GCS-HS steady-state mRNA levels on chemical exposure. Thus, either negative or positive modulation of steady-state
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., 1995
) 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., 1997). 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., 1999
). 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., 1993
), reduced levels of bcl-2 often reflect apoptogenic action (Mirkes et al., 1997
). 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 (50100 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., 1995; Toutain et al., 1998
). 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., 1995
; Toutain et al., 1998
), 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., 1995). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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