Potential conflict of interest: Nothing to report.
The potential for organ damage after using drugs or chemicals is a critical issue in medicine. To delineate mechanisms of drug-induced hepatic injury, we used transplanted cells as reporters in dipeptidyl peptidase IV–deficient mice. These mice were given phenytoin and rifampicin for 3 days, after which monocrotaline was given followed 1 day later by intrasplenic transplantation of healthy C57BL/6 mouse hepatocytes. We examined endothelial and hepatic damage by serologic or tissue studies and assessed changes in transplanted cell engraftment and liver repopulation by histochemical staining for dipeptidyl peptidase IV. Monocrotaline caused denudation of the hepatic sinusoidal endothelium and increased serum hyaluronic acid levels, along with superior transplanted cell engraftment. Together, phenytoin, rifampicin, and monocrotaline caused further endothelial damage, reflected by greater improvement in cell engraftment. Phenytoin, rifampicin, and monocrotaline produced injury in hepatocytes that was not apparent after conventional tissue studies. This led to transplanted cell proliferation and extensive liver repopulation over several weeks, which was more efficient in males compared with females, including greater induction by phenytoin and rifampicin of cytochrome P450 3A4 isoform that converts monocrotaline to toxic intermediates. Through this and other possible mechanisms, monocrotaline-induced injury in the endothelial compartment was retargeted to simultaneously involve hepatocytes over the long term. Moreover, after this hepatic injury, native liver cells were more susceptible to additional pro-oxidant injury through thyroid hormone, which accelerated the kinetics of liver repopulation. Conclusion: Transplanted reporter cells will be useful for obtaining insights into homeostatic mechanisms involving liver cell compartments, whereas targeted injury in hepatic endothelial and parenchymal cells with suitable drugs will also help advance liver cell therapy. (HEPATOLOGY 2007.)
To obtain fresh paradigms in tissue homeostasis after exposure to drugs or chemicals,1we considered that reporter cells will help elucidate perturbations in the liver, because genetically marked reporter cells can be inserted into liver compartments, including the parenchyma or hepatic endothelium.2–4 It was noteworthy that transplanted cells did not proliferate in the normal liver, where cell turnover is minimal, although, depending on the extent of injury in native cells, proliferation in healthy transplanted cells was activated.5, 6 In this way, transplanted cells could repopulate the liver, where in specific situations the kinetics of liver repopulation reflected loss of native hepatocytes.7–9 In healthy animals, genotoxic manipulations were particularly effective in promoting such liver repopulation with healthy cells. However, further insights are necessary to obtain pharmacologic approaches for repopulating the liver, particularly for clinical applications.
To develop cell compartment–specific hepatic perturbations, we used monocrotaline (MCT), a naturally occurring pyrrolizidine alkaloid with effects on the vascular endothelium,10–12 in combination with phenytoin and rifampicin, which are widely used in humans. These drugs offered potential for unique interactions as phenytoin and rifampicin activate complex cellular events, including oxidative stress, DNA damage, and cytochrome P450 (CYP) enzymes, e.g., CYP3A4, which converts MCT to reactive metabolites.13–16 Previously, we developed suitable mechanisms in mutant rats lacking dipeptidyl peptidase IV enzyme activity (DPPIV− rats) to determine the fate of transplanted cells.2, 3, 5–7 To determine whether transplanted cells could be used as reporters to identify changes in liver cell compartments, we used DPPIV− mice as recipients of healthy cells. Our studies established that transplanted reporter cells effectively identified perturbations in the hepatic endothelial and parenchymal cell compartments. Moreover, the combination of MCT, phenytoin, and rifampicin produced significant long-lasting changes in the liver that initially benefited transplanted cell engraftment and later resulted in the induction of proliferation in transplanted cells leading to extensive liver repopulation over time.
ANOVA, analysis of variance; CCl4, carbon tetrachloride; CYP, cytochrome P450; DPPIV, dipeptidyl peptidase IV enzyme; EM, electron microscopy; MCT, monocrotaline.
Materials and Methods
Phenytoin and rifampicin (Sigma Chemical Co., St. Louis, MO) were dissolved in 20 M NaOH and MCT in normal saline with pH to 7.4. After dose-ranging studies, mice received 30 mg/kg phenytoin and 75 mg/kg rifampicin intraperitoneally daily for 3 days. MCT was given as a single 200-mg/kg dose intraperitoneally on the 4th day. Cells were transplanted 24 hours after MCT. Other drugs were 0.71% sodium valproate in drinking water, 0.5 mL/kg carbon tetrachloride (CCl4) in olive oil (1:15, vol/vol) intraperitoneally, 8 mg/kg thyroxine in normal saline intraperitoneally (Sigma), and 0.3% cholic acid in rodent chow (Harlan Teklad Test Diets, Madison, WI).
DPPIV− mice in C57BL/6 background were 6 to 8 weeks old when used and were originally provided by Dr. D. Marguet (INSERM, Marseilles, France).17 Cell donors were 8-week-old to 15-week-old C57BL/6 mice from the National Cancer Institute (Bethesda, MD). Hepatocytes were isolated by 2-step collagenase perfusion, and 2 × 106 viable cells were injected immediately in 0.2 mL RPMI 1640 medium into the splenic pulp as described.4 The Animal Care and Use Committee at Albert Einstein College of Medicine approved protocols in compliance with National Research Council guidelines (Guide for the Care and Use of Laboratory Animals; United States Public Health Service publication, revised 1996).
Data were obtained from multiple mice in each group using 3 to 4 sections per animal. Formalin-fixed tissue sections of 10-μm to 15-μm thickness were stained with hematoxylin-eosin. To assess endothelial injury by electron microscopy (EM), liver was perfused through the portal vein from a 60-cm height with 3 to 4 mL 0.144 mol/L cacodylate buffer followed by 10 mL 1.5% glutaraldehyde in cacodylate buffer. After further incubation in glutaraldehyde, tissues were embedded in resin and stained with orcein. Ultrathin sections were examined under a JOEL electron microscope (Olympus, Tokyo, Japan). Endothelial integrity was graded under 1000× magnification in 50 consecutive sinusoids per mouse (n = 3) as follows: no injury (<25% sinusoid without endothelium); incomplete injury (25%-70% loss of endothelium); complete injury (no endothelial lining).
For apoptosis, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling was used (ApopTag Peroxidase In Situ Apoptosis Detection Kit, Chemicon International Inc., Temecula, CA). Briefly, 5-μm cryosections were fixed in 1% paraformaldehyde in phosphate-buffered saline, pH 7.4 for 10 minutes and post-fixed in ethanol:acetic acid 2:1 (vol/vol) for 5 minutes at −20°C. Positive control tissues were incubated with 100 units/mL deoxyribonuclease (DNAse) (Worthington Biochemical Corp., Lakewood, NJ) for 10 minutes. Tissues were quenched in 3% H2O2 in phosphate-buffered saline for 5 minutes, incubated with terminal deoxynucleotidyl transferase for 1 hour at 37°C followed by anti-digoxigenin-peroxidase for 30 minutes and color development using diaminobenzidine (DAB+ kit, DAKO Cytomation, Carpinteria, CA).
For Ki67 staining, sections were fixed in paraformaldehyde, blocked with 3% goat serum in phosphate-buffered saline containing 0.1% Triton X-100 for 1 hour, followed by incubation with rabbit anti-Ki67 (1:1000, Novocastra, Newcastle-upon-Tyne, UK) and peroxidase-conjugated anti-rabbit immunoglobulin G (1:300, Sigma) for 1 hour each followed by DAB.
Assays for Hepatocyte Turnover.
To identify DPPIV activity, 5-μm cryosections were fixed in chloroform-acetone and analyzed as described previously.2 Transplanted cell numbers per liver lobule and per unit liver volume (mm3) were quantitated as described with scoring under 400× of 100 consecutive lobules per liver lobe per animal using multiple sections.18 The extent of liver repopulation with transplanted cells was measured by ImageJ software (NCI, Bethesda, MD) in images taken under 40× (Spot RT digital camera, Diagnostic Instrument Inc., Sterling Heights, MI).
Tissue samples were dounce-homogenized in 0.25 M sucrose, 10 mM Tris hydrochloride, pH 7.5, and protease inhibitor cocktail (Calbiochem 539134, EMD Biosciences Inc., San Diego, CA). The lysate was centrifuged under 2000g for 25 minutes and then under 100,000g for 1 hour at 4°C. The pellet was sonicated, and 75 μg total proteins were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After transfer, nitrocellulose membranes were incubated for 1 hour with anti-CYP3A4 (1:4000, A4100; Xenotech, Lenexa, KA) and peroxidase-conjugated anti-rabbit immunoglobulin G (1:5000, 321804, Amersham Biosciences, Piscataway, NJ) for 1 hour with enzymatic chemiluminescence. Transblots were stained with Ponceau Red to visualize proteins. For immunostaining, 5-μm cryosections were fixed in 4% paraformaldehyde for 5 minutes at 4°C and blocked with 5% goat serum for 30 minutes. Anti-CYP3A4 (1:250) was applied for 1 hour, peroxidase-conjugated anti-rabbit immunoglobulin G (1:700, Sigma) was added for 30 minutes, and color was developed with DAB.
Alanine aminotransferase and total serum bilirubin were measured with an automated clinical system. Hyaluronic acid was measured with a commercial kit (Corgenix, Inc., Westminster, CO).19
Data are presented as means ± standard deviation. The significances were analyzed by Student t test, Kruskall-Wallis analysis of variance (ANOVA), or ANOVA on ranks using Tukey test for pairwise comparisons of mean group ranks. P < 0.05 was taken as significant.
Each animal group used 3 to 5 mice per time point. Initial studies using serum hyaluronic acid, light microscopy, EM, and liver tests indicated that 200 mg/kg MCT produced endothelial injury. After dose-ranging studies, we used 30 mg/kg phenytoin and 75 mg/kg rifampicin. To study combined drug effects, we used phenytoin and rifampicin for 3 days before MCT was given. Cells were transplanted 1 day after drugs (Fig. 1). We assessed cell engraftment after 7 days. To elucidate latent hepatic injury and its effects on liver cell turnover, we studied liver repopulation after 1, 2, 3, and 6 months. Typically, animals were treated with MCT alone, phenytoin or rifampicin plus MCT, or phenytoin plus rifampicin plus MCT. To establish drug dose–dependent changes, we used 50, 75, or 100 mg/kg rifampicin with fixed doses of phenytoin and MCT, followed by liver repopulation analysis. Gender-specific drug toxicity was defined in female mice. To reveal further perturbations, we used CCl4, cholic acid, valproic acid, and thyroxine, as indicated.
Characterization of Early Changes.
EM and serum hyaluronic acid levels showed that MCT rapidly damaged liver sinusoidal endothelial cells (Fig. 2). However, hepatocyte injury was not obvious in MCT-treated mice with light microscopy, EM, or blood tests (Table 1). Phenytoin plus rifampicin plus MCT produced only transient elevations in serum alanine aminotransferase, although liver histology and liver tests were normal over up to 2 months (Fig. 3).
Table 1. Changes in Liver Tests
Time After Treatments
Serum ALT (U/L)
Total Serum Bilirubin (mg/dL)
Phen+ Rif+ MCT
Phen+ Rif+ MCT
Normal control mice: serum ALT, 43 ± 2 U/L; total serum bilirubin, 0.30 ± 0.06 mg/dL.
P value <0.05 versus normal control mice, ANOVA on ranks with Tukey Test.
Further studies showed limited hepatic apoptosis after phenytoin plus rifampicin plus MCT, with only 4 to 8 apoptotic cells per high-power field (×400) after 4 or 8 weeks (Fig. 4).
Similarly, only infrequent hepatocytes expressed the proliferation marker Ki67 (Fig. 5).
Early Liver Changes and Cell Engraftment.
After MCT alone, engraftment of transplanted hepatocytes improved, reflecting superior entry of transplanted cells through disrupted endothelium into liver parenchyma (Fig. 6A,B). Phenytoin and rifampicin further improved cell engraftment, indicating additional endothelial injury (Fig. 6C-E). In drug-untreated mice, we observed 1.7 ± 0.1 transplanted cells per liver lobule and 753 ± 52 transplanted cells per cubic millimeter liver (Fig. 6F). In MCT-treated mice, 1.7-2-fold more transplanted cells were observed, P < 0.001, t tests. After phenytoin or rifampicin plus MCT, cell engraftment increased by up to 3-fold, P < 0.05, ANOVA.
Delayed Changes in Liver Cells.
In mice treated with either no drugs or MCT alone, transplanted cell numbers were unchanged over 3 months (Fig. 7A), indicating that native hepatocytes were not damaged. By contrast, in phenytoin plus MCT–treated mice, small proliferating foci of transplanted cells appeared after 2 and 3 months (Fig. 7B). This became more pronounced in rifampicin plus MCT–treated mice (Fig. 7C). In mice treated with phenytoin plus rifampicin plus MCT, transplanted cell proliferation was accelerated with significant liver repopulation after 1 month (Fig. 7D), indicating that despite discrete exposures to drugs, native hepatocytes were perturbed in a long-lasting and profound manner.
In male mice, the extent of liver repopulation 3 months after cell transplantation was as follows: MCT alone, <1%, phenytoin plus MCT, 9% ± 2%, rifampicin plus MCT, 21% ± 5%, and phenytoin plus rifampicin plus MCT, 46% ± 12%, P < 0.05, ANOVA (Fig. 8A). After 6 months, liver repopulation in phenytoin plus rifampicin plus MCT–treated mice increased to 62% ± 4%. Plotting of data using linear regression showed that liver repopulation to 50% took approximately 4 months in phenytoin plus rifampicin plus MCT–treated mice, reflecting equivalent losses of native cells during that period (Fig. 8B). This liver repopulation kinetics was 2-fold greater than that of rifampicin plus MCT–treated mice and 5-fold greater than that of phenytoin plus MCT–treated mice, reflecting higher attrition rates in native cells.
To demonstrate the effect of larger amounts of rifampicin, since this drug induced greater transplanted cell proliferation than phenytoin, we used 50, 75, and 100 mg/kg rifampicin with fixed doses of 30 mg/kg phenytoin and 200 mg/kg MCT. Recipients of 100 mg/kg rifampicin exhibited 50% to 60% mortality even before cell transplantation, indicating greater acute toxicity. Rifampicin produced dose-dependent changes in liver repopulation after 2 months, which was more with 75 mg/kg (23% ± 7%) or 100 mg/kg rifampicin (23% ± 3%) compared with 50 mg/kg rifampicin (11% ± 7%), P < 0.05, ANOVA.
To demonstrate sex-specific mechanisms in hepatotoxicity, we treated female mice with 75 or 100 mg/kg rifampicin plus 30 mg/kg phenytoin plus 200 mg/kg MCT. Female mice were less susceptible to drug toxicity with no mortality despite 100 mg/kg rifampicin combination. This was verified by less liver repopulation in female mice (Fig. 8C); 18-fold less, compared with male mice treated with 75 mg/kg rifampicin after 3 months, P < 0.001, t test (Fig. 8A). In recipients of phenytoin plus rifampicin, hepatic CYP3A4 expression was induced as demonstrated by immunostaining and western blotting (Fig. 8D-G).
Drug-Treated Liver Was Susceptible to Further Perturbations.
Treatment first with phenytoin plus rifampicin plus MCT and then CCl4 produced injury in native, as well as transplanted cells in middle and perivenous zones of the liver lobule (Fig. 9B). Consequently, liver repopulation decreased to only 14% after 2 months, approximately 50% of that in CCl4-untreated control mice. Also, the toxic bile salt cholic acid produced injury in native and transplanted cells, leading to decreased liver repopulation of only 7% after 2 months. Similarly, liver repopulation declined to 5% after valproic acid after 2 months (Fig. 9C). However, treatment with thyroxine significantly enhanced transplanted cell proliferation, suggesting selective oxidative injury in native cells previously exposed to phenytoin, rifampicin, and MCT (Fig. 9D,E).
These studies established that transplanted reporter cells effectively elicited damage in hepatic endothelial and parenchymal compartments. Improvement in transplanted cell engraftment verified onset of endothelial damage, as this benefits entry of transplanted cells into the liver parenchyma.20 Analysis of transplanted cell proliferation, which requires loss of native cells, showed that the liver had undergone changes, which were not demonstrated by conventional tissue analysis. Recently, unexpected drug toxicity was encountered after use of even single drugs, for example, liver failure with the antiviral drug fialuridine,21 the anti-diabetic drug troglitazone, and the anti-thrombotic drug, ximelagatran.22 Therefore, despite innovative applications of toxicogenomic, proteomic, metabolonomic, and in vitro assays for assessing mechanisms of drug toxicity, further approaches are needed.
Absence of transplanted cell proliferation after MCT alone indicated that phenytoin and rifampicin contributed to hepatic injury. Interactions between MCT, phenytoin, and rifampicin were at multiple levels, including CYP3A4 regulation by phenytoin and rifampicin, as shown here, through regulation of other CYP isoforms by phenytoin and rifampicin, and also by MCT itself, as pyrrolizidine alkaloids regulate CYP2E1 and other CYPs.13, 14, 23 Also, sex-specific mechanisms altered drug toxicity in this model, with phenytoin and rifampicin inducing CYP3A4 less in female mice. However, CYP3A4 induction was less than 2-fold different between male and female mice, whereas liver repopulation (or native liver cell turnover) was 18-fold different. Therefore, additional mechanisms, such as direct genotoxicity, pro-oxidant damage, or even protection by estrogens, may have been involved.12
The ability of transplanted cells to proliferate in mice treated with MCT, phenytoin, and rifampicin indicated sparing of these cells from secondary processes, such as recycling of toxic metabolites released by damaged or dying cells. The ability of transplanted cells to retain drug metabolic activity will be significant given that greater expression of specific CYPs makes perivenous hepatocytes more susceptible to toxins.6, 24 As transplanted cells engraft in periportal areas of the liver lobule, in contrast to perivenous areas, this protects transplanted cells from perivenous toxins. However, during liver repopulation, when transplanted cells enter midzonal or perivenous areas of the lobule, gene expression patterns switch to match that of adjacent native hepatocytes. Therefore, CCl4-induced loss of transplanted cells should not be surprising in view of its metabolism in perivenous and midzonal areas of the liver. Similarly, toxic bile salts potentiate hepatic apoptosis and accelerate liver repopulation in the setting of abnormal biliary transport.25, 26 In our studies, decreased liver repopulation in mice treated with cholic acid indicated that both native and transplanted hepatocytes were affected. Similarly, nonselective damage in native and transplanted hepatocytes likely accounted for decreased liver repopulation after valproic acid, which causes cell injury through pro-oxidant and other mechanisms.27 Conversely, thyroxine administration to mice treated with phenytoin, rifampicin, and MCT accelerated liver repopulation, which was in agreement with the ability of thyroid hormone to stimulate hepatic DNA synthesis while promoting apoptosis in cells with genotoxic damage.28, 29
Taken together, these findings indicated that in mice exposed to phenytoin, rifampicin, and MCT, liver injury was age-dependent and gender-dependent, as shown here with 6-week-old to 8-week-old male mice. We used cell donors of 8 weeks' to 15 weeks' age for convenience; their age should have been immaterial unless aging-associated genetic lesions were to alter replication in healthy donor cells, which has not been shown. We consider that the central mechanism responsible for liver repopulation in MCT-treated mice involves loss of native cells, thereby requiring healthy transplanted cells to proliferate. Although cell transplantation in the liver transiently activates Kupffer cells, stellate cells and endothelial cells with hepatic expression of trophic, for example, hepatocyte growth factor, vascular endothelial growth factor, matrix-remodeling, metalloproteinases, and other genes that affect cell engraftment,2, 3, 30, 31 whether transplanted cell proliferation during liver repopulation is regulated by specific factors released by liver cells is unknown. Nonetheless, because transplanted cells retain physiologically regulated function after liver repopulation,6, 7 including in animal models of disease,9, 26, 32 our findings should offer impetus for drug-based strategies to damage the native liver before cell transplantation. This should facilitate studies of cell and gene therapy, stem/progenitor cells, as well as animal model development.