Department of Pharmacology and Toxicology, B-346 Life Sciences Building, Michigan State University, East Lansing, Michigan 48824
Received August 1, 2001; accepted October 19, 2001
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ABSTRACT |
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Key Words: monocrotaline; liver; rat; fibrin deposition; sinusoidal endothelial cell.
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INTRODUCTION |
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MCT is an unreactive compound that requires bioactivation by cytochromes P450 to produce hepatic and pulmonary toxicity (Schultze and Roth, 1998; Wilson et al., 1992
). Monocrotaline pyrrole (MCTP), also called dehydromonocrotaline, is a dehydrogenation product of MCT produced in the liver (Glowaz et al., 1992
; Pan et al., 1993
). This metabolite is thought to be responsible for the toxicity of MCT in vivo (Butler et al., 1970
; Lafranconi and Huxtable, 1984
; Pan et al., 1993
; Roth and Reindel, 1990
).
In the liver, MCT produces centrilobular parenchymal cell necrosis, endothelial cell damage (both central venular and sinusoidal), congestion and dilation of the sinusoids, and hemorrhage (DeLeve et al., 1999; Schoental and Head, 1955
; Wang et al., 2000
). Histopathological examination of liver sections by light microscopy revealed central venular endothelial cell (CVEC) damage in MCT-treated rats (DeLeve et al., 1999
; Schoental and Head, 1955
; Wang et al., 2000
). These lesions may progress to complete disruption of vascular intima. Treatment of rats with MCT also damages endothelial cells lining sinusoids (SECs; DeLeve et al., 1999
). Toxicity to SECs is progressive, beginning with defenestration and gap formation in the SEC lining and culminating in complete loss of SECs from the sinusoid (DeLeve et al., 1999
). Although these studies have clearly shown that SEC and CVEC damage occurs in the liver, they have not addressed the severity and distribution of SEC toxicity or delineated the onset of endothelial cell injury.
One consequence of endothelial cell damage in the vasculature is activation of the coagulation system (Colman et al., 1994; Schultze and Roth, 1998
). During vascular injury, the anticoagulant and profibrinolytic properties of endothelial cells are lost, and the coagulation cascade is initiated by the tissue factor pathway. The series of blood zymogens comprising the coagulation cascade becomes activated and promotes platelet activation and fibrin deposition. In addition, loss of endothelial cells from the vasculature can expose coagulation factors to negatively charged subendothelial surfaces that can catalyze their activation. Since endothelial cell damage occurs in the liver after MCT treatment, it is probable that the coagulation system becomes activated. Several groups have reported that fibrin deposits in the liver after MCT treatment (Butler et al., 1970
; Schoental and Head, 1955
); however, others have not observed fibrin deposition (DeLeve et al., 1999
). These differences might be related to differences in dose, route of administration, and the time at which livers were examined. Previous studies in which fibrin deposition in the liver has been observed after MCT treatment were qualitative, and they did not evaluate the extent or zonal distribution. In addition, the temporal relationship between fibrin deposition and the onset of hepatic parenchymal cell injury has not been reported. This may be relevant to the mechanism of injury because in a number of models of liver injury, coagulation system activation is required for hepatic parenchymal cell injury (Arai et al., 1996
; Fujiwara et al., 1988
; Hewett and Roth, 1995
; Yamada et al., 1989
).
The studies presented herein tested the hypothesis that MCT produces zone-specific damage to endothelial cells and fibrin deposition in the liver. To this end, morphometric analysis of endothelial cells and fibrin in livers from MCT-treated rats was conducted using immunohistochemical techniques. The results of these studies show that treatment of rats with MCT produces time-dependent injury to SECs and CVECs that precedes parenchymal cell injury and only occurs in centrilobular regions of the liver lobule, i.e., regions that develop parenchymal cell necrosis. Similarly, fibrin deposition occurred only in centrilobular regions after MCT treatment and preceded the onset of hepatic parenchymal cell injury.
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MATERIALS AND METHODS |
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Treatment protocol.
Rats were fasted for 24 h prior to treatment with MCT (Trans World Chemicals, Inc., Rockville, MD). They received MCT by ip injection at doses indicated in the text and figures or an equivalent volume of sterile saline vehicle. Food was returned to the rats after MCT treatment. MCT was dissolved in sterile saline, minimally acidified by 2M HCl. The pH was brought to 6.7 by addition of 4M NaOH, and the volume was adjusted with sterile saline to the appropriate final concentration.
Assessment of hepatic injury, plasma fibrinogen, and plasma hyaluronic acid.
At 4, 8, 12, or 18 h after treatment with MCT or its vehicle, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip). A midline abdominal incision was made, and blood was collected from the descending aorta into a syringe containing sodium citrate (final concentration, 0.38%). Hepatic parenchymal cell injury was evaluated by measuring the activity of alanine aminotransferase (ALT) in the plasma using Sigma kit 59-UV (Sigma Chemical Co., St. Louis, MO). One transverse section from the middle of the left lateral liver lobe and one from the right lateral lobe were processed for light microscopy. Paraffin-embedded sections were cut at 5 µm, stained with hematoxylin and eosin (H & E), and evaluated using a light microscope. Another portion of the liver was frozen for immunohistochemical staining as described below. The remaining liver was snap frozen in liquid nitrogen for evaluation of tissue hemoglobin as described below. Plasma fibrinogen concentration was determined from the thrombin clotting time of diluted samples by using a fibrometer and a commercially available kit (Sigma Kit 886-A). Plasma hyaluronic acid was measured using a commercially available, enzyme-linked immunosorbent assay (ELISA; Chugai Diagnostics Science Co., Tokyo, Japan).
Liver hemoglobin.
The concentration of liver hemoglobin was used as a biomarker of hemorrhage and was estimated using a commercially available kit (Total Hemoglobin Kit; Sigma Chemical Co.) as described (Jaeschke et al., 2000). A 20% homogenate was made from samples of frozen liver in 50 mM sodium phosphate buffer (120 mM NaCl, 10 mM EDTA). The samples were centrifuged at 16,000 x g for 10 min at 4°C, and 200 µl of supernatant was diluted in Drabkin's solution. After a 15-min incubation, the absorbance was measured at 540 nm, and the concentration of hemoglobin was calculated from a standard curve.
Immunohistochemistry.
A 1 cm3 block of liver cut from the middle of the left lateral liver lobe was frozen for 8 min in isopentane immersed in liquid nitrogen. For liver endothelial cell immunostaining, 8 µm-thick sections of frozen liver were fixed in acetone (4°C) for 5 min. Next, they were incubated with PBS containing 10% goat serum (i.e., blocking solution; Vector Laboratories, Burlingame, CA) for 30 min, then with mouse antirat RECA-1 (rat endothelial cell antigen-1, Serotec, Inc., Raleigh, NC), diluted (1:20) in blocking solution overnight at 4°C. The RECA-1 antibody binds to rat endothelium but not other cell types (Duijvestijn et al., 1992). In the liver, this antibody stains both SECs and endothelial cells of larger vessels. After incubation with the RECA-1 antibody, sections were incubated for 3 h with goat antimouse secondary antibody conjugated to Alexa 594 (1:1000, Molecular Probes, Eugene, OR) in blocking solution containing 2% rat serum. Sections were washed 3 times, 5 min each, with PBS and visualized using a fluorescent microscope.
For fibrin immunostaining, 8 µm-thick sections of frozen liver were fixed in 10% buffered formalin containing 2% acetic acid for 30 min at room temperature. This fixation protocol solubilizes all fibrinogen and fibrin species except for cross-linked fibrin; therefore, only cross-linked fibrin stains in sections of liver (Schnitt et al., 1993). Sections were blocked with PBS containing 10% horse serum (i.e., blocking solution; Vector Laboratories) for 30 min, and this was followed by incubation overnight at 4°C with goat antirat fibrinogen diluted (1:1000, ICN Pharmaceuticals, Aurora, OH) in blocking solution. Next, sections were incubated for 3 h with donkey antigoat secondary antibody conjugated to Alexa 594 (1:1000, Molecular Probes) in blocking solution for 3 h. Sections were washed 3 times, 5 min each, with PBS and visualized using a fluorescent microscope.
For both protocols, no staining was observed in controls in which the primary or secondary antibody was eliminated from the staining protocol. All treatment groups that were compared morphometrically were immunohistochemically stained at the same time.
Morphometric quantitation of endothelial cells and fibrin in the liver.
Endothelial cells and fibrin deposition in the liver were quantified morphometrically by analyzing the area of immunohistochemical staining in a section of liver. A decrease in the area of staining for endothelial cells suggests a loss of these cells in the liver. An increase in the area of staining of fibrin in the liver indicates fibrin deposition. These were quantified in 2 ways. First, morphometric analysis was performed to quantify the area of endothelial cell or fibrin staining in randomly chosen, low-power fields irrespective of the region of the liver (i.e., centrilobular, periportal, etc.). This was done to determine if the total area of endothelial cells or fibrin deposition in sections of liver changed after MCT treatment. Secondly, the area of endothelial cells or fibrin deposition was determined in randomly chosen, centrilobular and periportal regions separately. This was done to determine if zonal differences in staining occurred after MCT treatment.
Fluorescent staining in sections of liver was visualized on an Olympus AX-80T microscope (Olympus, Lake Success, NY). For morphometric analysis of the total area of endothelial cells or fibrin deposition in a liver section, digital images of 10, randomly chosen, low power (magnification x100) fields per tissue section were captured using a SPOT II camera and SPOT advanced software (Diagnostic Instruments, Sterling Heights, MI). Samples were coded such that the evaluator was not aware of the treatment, and the same exposure time was used for all captured images. Each digital image encompassed a total area of 1.4 mm2 and contained several centrilobular, periportal, and midzonal regions. For morphometric analysis of the area of staining within centrilobular or periportal regions, digital images of 5 randomly chosen fields (magnification x200) that contained a centrilobular region and 5 randomly chosen fields that contained a periportal region were captured. The same exposure time was used to capture all images.
The area of immunohistochemical staining (number of pixels) was quantified using Scion Image software (Scion Corporation, Frederick, MD). For endothelial cell quantification, a density slice was taken from an inverted, gray-scale digital image of a liver section from a saline-treated rat. A density slice allows analysis of pixels in a defined range of gray values (i.e., densities). Next, the threshold was adjusted such that background staining was eliminated from the analysis and only endothelial cell staining was counted. The same threshold value was used to analyze digital images from sections from all treatment groups. For quantification of fibrin deposition, the threshold was selected so that little positive staining was present in saline-treated controls. The same threshold value was used to analyze digital images from all treatment groups. For quantification of endothelial cells or fibrin in a low power field of liver, the area of positive staining was measured and divided by the total area of the image.
For analysis of endothelial cells or fibrin deposition in centrilobular or periportal regions, a 145 µm-diameter circle was drawn around the central vein or the periportal region. The circumference of the circle was approximately 45 hepatocytes away from the central vein or portal region. This area was arbitrarily defined as the centrilobular or periportal region. The total area analyzed was 16,512 µm2. The area of endothelial cell or fibrin staining in that region was measured as described above and divided by the area of the circle.
For both, the staining is expressed as a fraction of the total area. The random fields analyzed for each liver section were averaged and counted as a replicate, i.e., each replicate represents a different rat. For the time-course studies, saline-treated rats at various time-points were combined into 1 group for statistical analysis since no differences occurred among saline-treated groups.
Statistical analysis.
Results are presented as the mean ± SEM. A 1-way, completely random ANOVA was used to analyze time-dependent changes in plasma ALT, plasma fibrinogen, plasma hyaluronic acid, liver RECA-1 staining, liver fibrin staining, and liver hemoglobin concentration. Comparisons among group means were made using the Student-Newman-Keuls test. Data from studies comparing RECA-1 staining and fibrin staining in periportal and centrilobular regions were analyzed using a 2 x 2 multifactorial, completely random ANOVA. ANOVAs were performed on log-transformed data in instances in which variances were not homogeneous. Data expressed as a fraction were transformed by arc sine square root prior to analysis. Comparisons among group means were made using the Student-Newman-Keuls test. The criterion for significance was p < 0.05 for all studies.
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RESULTS |
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DISCUSSION |
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To whom correspondence should be addressed.Previous studies have shown by light and electron microscopy that damage occurs to both CVECs and SECs within the liver after MCT treatment (Allen et al., 1969; Brooks et al., 1970
; Butler et al., 1970
; DeLeve et al., 1999
). In addition, studies in vitro indicate that MCT directly damages SECs, suggesting that these cells may be a target for toxicity in the liver (DeLeve et al., 1996
). It is unclear from these studies, however, whether MCT produces widespread endothelial cell damage in the liver or whether it is limited to zonal or focal lesions. In the studies presented here, morphometric quantitation of immunohistochemically stained liver sections revealed that endothelial cell damage was extensive. On average, approximately 40% of the endothelial cell staining in the liver was completely lost within 12 h after MCT treatment (Figs. 4 and 5
). In many regions, the sinusoids and central veins were completely denuded of endothelial cells (Fig. 4
). This is consistent with what has been described previously using electron microscopic analysis of liver tissue from rats, monkeys, and humans exposed to MCT; however, the studies presented here are the first to quantify endothelial cell damage after MCT treatment and to define its time of onset (Allen et al., 1969
; Brooks et al., 1970
; DeLeve et al., 1999
).
The antigen that the RECA-1 antibody recognizes has not been determined (Duijvestijn et al., 1992), and it is possible that expression of this antigen was modified by MCT. To confirm that the loss of RECA-1 staining was due to detachment and loss of endothelial cells and not due to down-regulation of the RECA-1 antigen, electron microscopy was performed on the same tissues that were analyzed by immunohistochemistry in Figure 4
. Electron microscopy confirmed that SECs were absent in many sinusoidal regions 18 h after treatment with 300 mg/kg MCT (data not shown), which suggested that the decrease in RECA-1 staining was due to endothelial cell loss and not to down-regulation of the RECA-1 antigen. In addition, the increase in plasma hyaluronic acid observed after MCT treatment (Fig. 3
) supports the immunohistochemical data suggesting endothelial cell injury.
Interestingly, severe endothelial cell injury leading to a complete detachment of SECs and CVECs occurred only in centrilobular regions (Figs. 4 and 5). In livers of humans exposed to PAs, damage to sinusoidal endothelial cells occurred in all regions of the liver lobule (i.e., periportal, midzonal, centrilobular; Brooks et al., 1970
). Occasional extravasation of erythocytes into the space of Disse in the periportal region were observed by electron microscopy in the studies presented here (data not shown), suggesting that changes to the sinusoidal endothelium do occur in this region. However, these changes were much less than those observed in the centrilobular region and likely too subtle to be reflected in changes in RECA-1 staining. In addition, it is apparent from the RECA-1 immunohistochemical staining (Fig. 4
) that loss of SECs occurred in the midzonal region, although this area was not quantified due to the difficulty of defining this region in tissue sections for morphometric analysis. Accordingly, SEC damage occurred to some extent in all of the regions of the liver lobule early after MCT exposure; however, damage to centrilobular endothelium was most severe, and damage in other regions was subtle.
The mechanisms contributing to greater endothelial cell toxicity in the centrilobular compared to the periportal region are not known, but they may be related to differences in the extent of metabolic activation or detoxification of MCT in these two zones. MCT requires bioactivation by cytochromes P450 to produce liver injury (Schultze and Roth, 1998; Wilson et al., 1992
), and SECs express cytochromes P450 (Lester et al., 1993
). Studies in vitro have suggested that SECs can bioactivate MCT to a toxic metabolite (DeLeve et al., 1996
). The observation that greater SEC injury occurred in centrilobular regions suggests the possibility of zonal differences in expression of cytochromes P450 in SECs, similar to what has been observed in parenchymal cells (Oinonen and Lindros, 1998
). Cytochromes P450 of the 3A family metabolize MCT to MCTP (Kasahara et al., 1997
), and members of this P450 family are primarily expressed in centrilobular regions (Oinonen and Lindros, 1998
). Accordingly, if SECs express these, activity may be greater in centrilobular SECs. Differences in zonal distribution of SEC proteins have been reported, suggesting that gene regulation differences exist in SECs from different zones of the liver lobule (Scoazec et al., 1994
). Alternatively, it is possible that metabolism of MCT to MCTP by parenchymal cells contributes to SEC toxicity in vivo. That is, injury to centrilobular SECs might be greater because cytochromes P450 of the 3A family are primarily expressed by parenchymal cells of this region. Finally, the difference in zonal toxicity observed in vivo might result from an inability of centrilobular SECs to detoxify MCTP. This is unlikely, because injection of MCTP preferentially damaged endothelium of periportal regions (Butler et al., 1970
). This result is inconsistent with periportal SECs being more capable of detoxifying MCTP and suggests that zonation of endothelial cell toxicity observed after MCT treatment may be more related to the location in which the MCTP is produced.
In vitro, SECs are more sensitive to the toxic effects of MCT than parenchymal cells (DeLeve et al., 1996). Accordingly, it has been suggested that MCT-induced damage to SECs in vivo results in microcirculatory disturbances that lead to hypoperfusion of the liver and consequent parenchymal cell injury (DeLeve et al., 1996
). Observations presented here support this hypothesis. First, the histopathological finding of coagulative, hepatocelluar necrosis in centrilobular regions is consistent with ischemic injury (Fig. 2
; DeLeve et al., 1999
). Second, if destruction of SECs contributes causally to microcirculatory changes and parenchymal cell damage, then it should precede these changes. The plasma concentration of hyaluronic acid began to increase between 4 and 8 h after MCT treatment (Fig. 3
), suggesting an early onset of endothelial cell injury. SEC injury was most severe in centrilobular regions (Fig. 5
) and preceded evidence of hemorrhage (i.e., liver hemoglobin, Fig. 6
) and parenchymal cell injury (Fig. 1
). Both of these changes occurred primarily in centrilobular regions and began between 8 and 12 h after treatment (Figs. 1 and 6
). Therefore, extensive SEC damage in centrilobular regions may lead to microcirculatory disturbances that promote ischemic, hepatic parenchymal cell injury in this region.
Another factor that may contribute to ischemic parenchymal injury after MCT treatment is fibrin deposition. Electron microscopic studies have shown that fibrin deposition occurs in the liver after MCT treatment (Allen et al., 1969; Butler et al., 1970
; Schoental and Head, 1955
). Fibrin deposition was not quantified in these early studies, and the temporal relationship between the appearance of fibrin and the onset of endothelial cell and hepatic parenchymal cell injury was not defined. Treatment of rats with 300 mg/kg MCT provoked activation of the coagulation system (Fig. 7
) and fibrin deposition in the liver (Fig. 9
). Fibrin deposition occurred simultaneously with endothelial cell injury and prior to hepatic parenchymal cell injury. In addition, it occurred only in centrilobular regions, where endothelial and hepatic parenchymal cell injury were extensive. This suggests the possibility of a causal link between fibrin deposition and hepatic parenchymal cell injury. Several hepatotoxicants promote coagulation system activation and fibrin deposition in the liver (Ahmed et al., 1987
; Arai et al., 1996
; Fujiwara et al., 1988
; Pearson et al., 1996
), and inhibition of coagulation system activation completely prevents hepatic parenchymal cell injury in these models (Arai et al., 1996
; Fujiwara et al., 1988
; Pearson et al., 1996
). This suggests that the coagulation system may be a critical mediator of liver injury after exposure to some hepatotoxicants. Considering the extent of fibrin deposition in the liver and the nature and location of the hepatic lesions, it is reasonable to hypothesize that coagulation activation and fibrin deposition are required for MCT-induced liver injury.
Fibrin deposition in the liver after exposure of rats or humans to MCT has not been a consistent finding (Brooks et al., 1970; DeLeve et al., 1999
). Electron microscopic analysis of liver samples from children exposed to PA-containing plants showed no evidence of fibrin deposition (Brooks et al., 1970
). However, these samples were not taken until several weeks after the children were exposed to PAs, and it is possible that within this time the fibrinolytic system had been activated and removed fibrin from the liver. Fibrin deposition may not occur in all models of MCT-induced liver injury. In a detailed study by DeLeve et al. fibrin deposition in the rat liver was not observed at any time after MCT treatment (DeLeve et al., 1999
). Clearly, further study is needed to understand the role of the coagulation system in MCT hepatotoxicity.
In summary, treatment of rats with MCT produced extensive endothelial cell injury and fibrin deposition in centrilobular regions of the liver. These events were followed by extensive hemorrhage and evidence of hepatic parenchymal cell injury. The results raise the possibility that MCT produces direct damage to centrilobular endothelial cells, which promotes hemorrhage and fibrin deposition in the liver. Such events might lead to local hypoperfusion that could contribute to parenchymal cell injury.
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ACKNOWLEDGMENTS |
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NOTES |
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