Toxicological Sciences 55, 444-452 (2000)
Copyright © 2000 by the Society of Toxicology
Bacterial Lipopolysaccharide Exposure Augments Aflatoxin B1-Induced Liver Injury
Department of Pharmacology and Toxicology, National Center for Food Safety and Toxicology and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824
Received November 9, 1999; accepted January 24, 2000
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bacterial endotoxin (lipopolysaccharide; LPS) given to animals in large doses results in pronounced, midzonal liver injury. Exposure to smaller, non-injurious doses of LPS augments the toxicity of certain hepatotoxicants. This study was conducted to delineate the development of injury in a rat model of augmentation of aflatoxin B1 (AFB1) hepatotoxicity by LPS. At large doses (i.e., > 1 mg/kg, ip), AFB1 administration resulted in pronounced injury to the periportal regions of the liver. Male, Sprague-Dawley rats (250–350 g) were treated with 1 mg AFB1/kg, ip or its vehicle (0.5% DMSO/saline) and 4 h later with either E. coli LPS (7.4 x 106 EU/kg, iv) or its saline vehicle. Liver injury was assessed 6, 12, 24, 48, 72, or 96 h after AFB1 administration. Hepatic parenchymal cell injury was evaluated as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum and from histologic examination of liver sections. Biliary tract alterations were evaluated as increased concentration of serum bile acids and activities of
-glutamyltransferase (GGT), alkaline phosphatase (ALP), and 5'-nucleotidase (5'-ND) in serum. At all times and for all markers, injury in rats treated with either AFB1 or LPS alone was absent or modest. In the AFB1/LPS cotreated group, hepatic parenchymal cell injury was pronounced by 24 h and had returned to control values by 72 h. The injury began in the periportal region and spread midzonally with time. Furthermore, changes in serum markers indicative of biliary tract alterations were evident by 12 h and had returned to control values by 72 h. Thus, the nature of the hepatic lesions suggested that LPS potentiated the effects of AFB1 on both parenchymal and bile duct epithelial cells. Key Words: aflatoxin B1; apoptosis; endotoxin; lipopolysaccharide; LPS; liver injury; necrosis; oncosis; sensitivity to intoxication; sepsis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aflatoxin B1 (AFB1) is a metabolite produced by the fungi, Aspergillus flavus and Aspergillus parasiticus, which are contaminants of human and animal grain foods. Corn is probably the most important source of AFB1 for both human and animal consumption (Wood, 1989
). In contrast to the USA, concentrations of AFB1 in human food in some developing countries are quite large. For example, the average daily intake of AFB1 in Guangxi Province of the People's Republic of China has been reported to be between 50 and 75 µg/day (Groopman et al., 1992, 1994).
AFB1 causes acute hepatotoxicity and liver carcinomas in humans and laboratory animals (Chao et al., 1991). It is metabolized by cytochrome P-450 monooxygenases to reactive aflatoxin B1-8,9-epoxide, which binds to cellular macromolecules and causes injury to periportal regions of the liver. This damage is evident acutely as hemorrhage, parenchymal cell necrosis, and injury to intrahepatic bile ducts. The liver lesions along with loss of appetite and lethargy in exposed animals are collectively referred to as aflatoxicosis.
Endotoxic lipopolysaccharide (LPS) is a constituent of the outer membrane of the cell walls of Gram-negative bacteria. It has been extensively studied as an agent of inflammation and a major contributing factor to the pathogenesis of bacterial infection. Striking parallels exist between the effects of LPS in experimental animals and those observed in patients with Gram-negative bacterial sepsis. Among the changes observed upon exposure to LPS are fever, circulatory shock, disseminated intravascular coagulation, and damage to numerous organs including the liver (Ghosh et al., 1993). Although the mechanisms contributing to tissue injury by LPS are many and may vary among tissues, a commonality appears to be the involvement of host-derived, soluble and cellular mediators of inflammation (Molvig et al., 1988). Interactions among several of these appear to be necessary for full manifestation of tissue injury during LPS exposure (Hewett and Roth, 1993). For example, at large doses LPS induces midzonal liver injury in rats, and this requires inflammatory mediators such as neutrophils (Hewett et al., 1992; Jaeschke et al., 1993), Kupffer cells (Arthur et al., 1986, 1985), TNF-
(Hewett et al., 1993), platelets (Pearson et al., 1995), and thrombin (Hewett and Roth, 1995; Moulin et al., 1996; Pearson et al., 1996).
Exposure to smaller doses of LPS initiates a more modest and noninjurious inflammatory response. Such LPS exposure can render the liver more sensitive to injury from hepatotoxic chemicals (Lind et al., 1984; Sneed et al., 1997; Taylor et al., 1991). Indeed, it has been suggested (Nolan, 1989) that exposure to endogenous LPS due to increased movement of bacteria across a compromised intestinal mucosa contributes to the hepatotoxicity produced by various agents, including, carbon tetrachloride (Nolan, 1975) and alpha-naphthylisothiocyanate (ANIT) (Calcamuggi et al., 1992). The mechanism behind this increased sensitivity has yet to be determined, but it is likely that aspects of the inflammatory response initiated by exposure to small amounts of LPS are responsible (Roth et al., 1997).
People are commonly exposed to LPS via translocation of it from the gastrointestinal (GI) lumen into the circulation as well as from bacterial infections. Clinical studies in humans have revealed that small amounts of LPS normally escape through the GI barrier and enter the circulation (Jacob et al., 1977; Nolan, 1981). Furthermore, systemic endotoxemia without bacterial infection has been reported in patients with liver or gastrointestinal diseases. Also, changes in diet or alcohol consumption can significantly increase plasma LPS concentration (Roth et al., 1997).
The evidence presented above indicates that (1) LPS exposure can augment the toxicity of certain chemicals, and (2) systemic exposure to LPS in people is common and varies considerably among and within individuals. It follows that LPS exposure might be an important determinant of susceptibility to intoxication from certain xenobiotics. This study was undertaken to determine if a small dose of LPS could augment the response to a toxin to which people are often exposed. The hypothesis tested was that LPS potentiates the acute hepatocellular and biliary effects of AFB1. To test this, the development of liver injury was evaluated in rats cotreated with doses of LPS and AFB1, which were non-injurious when given alone.
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MATERIALS AND METHODS |
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Animals and materials.
Male, Sprague-Dawley rats (CD-Crl:CD-(SD)BR VAF/Plus; Charles River, Portage, MI) weighing 250–350 g were used in these studies. The reagent kits used for measuring serum markers of liver injury (ALT, 59-UV; AST, 58-UV; GGT, 419; bile acids, 450; ALP, 245; and 5'-ND, 265-UV) were purchased from Sigma Chemical Co. (St. Louis, MO). Lipopolysaccharide derived from E. coli serotype 0128:B12 with an activity of 1.7 x 106 EU/mg was purchased from Sigma. A colorometric, kinetic Limulus Amebocyte Lysate (LAL) assay was employed to estimate LPS concentration using a kit (#50–650U) purchased from BioWhittaker (Walkersville, MD). Unless stated otherwise, all chemicals were purchased from Sigma Chemical Co.
Treatment protocol.
Rats fasted for 24 h were given 1 mg AFB1/kg or vehicle (0.5% DMSO in 0.9% sterile saline), ip, followed 4 h later by 7.4 x 106 EU LPS/kg or sterile saline via the tail vein. This AFB1/LPS cotreatment regimen was used because it was found in preliminary studies to result in hepatotoxicity, whereas the doses of AFB1 and LPS were non-injurious by themselves, as indicated by a lack of increase in serum markers of liver injury compared to vehicle controls. At 6, 12, 24, 48, 72, or 96 h after AFB1 administration, the rats were anesthetized with sodium pentobarbital (50 mg/kg, ip), and blood was drawn from the dorsal aorta, allowed to clot, and centrifuged to separate serum. Before the liver samples were placed in neutral buffered formalin, a midlobe radial section of the right anterior lobe was freeze-clamped in liquid nitrogen. For histological comparisons of AFB1/LPS cotreatment to treatment with a larger, injurious dose of each agent given alone, either AFB1 or LPS alone was administered. Specifically, rats fasted for 24 h were given 4 mg AFB1, ip, or 0.5 x 108 EU LPS/kg, iv. Twenty-four h after administration, they were anesthetized and serum samples and liver were taken. These large doses were chosen because preliminary studies demonstrated that they resulted in elevated serum ALT and AST activities 24 h after treatment.
Determination of Hepatotoxicity
Serum markers of liver injury.
Reagent kits (see Animals and materials) were used to measure serum markers of liver injury. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured spectrophotometrically by the methods of Wroblewski and LaDue (1956) and Karmen (1955), respectively. Serum glutamyltransferase (GGT), 5'-nucleotidase (5'-ND), and alkaline phosphatase (ALP) activities were measured by the methods of Szasz and Persijn (1974), Arkesteijn (1976), and Bowers and McComb (1966), respectively. Enzyme activities are expressed as International Units per liter of serum (U/L). Serum bile acids concentration was measured by the method of Mashige and co-workers (Mashige et al., 1981).
Histopathologic evaluation.
A midlobe, radial section of the right anterior lobe of the liver from each rat was fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were cut at 5 µm thickness and stained with hematoxylin and eosin (H&E). Slides were randomized, coded, and evaluated with light microscopy. Following the recommendations of the Society of Toxicologic Pathologists' Committee on the Nomenclature of Cell Death (Levin et al., 1999), this paper uses the term "necrosis" to describe cell death, regardless of the particular pathway on which cell death occurred. The terms "apoptosis" and "oncosis" are used to distinguish necrotic cells based on morphological characteristics described by the Committee. Oncosis was defined as swollen cells having pyknotic or karyolytic nuclei and hypereosinophilic cytoplasm. Apoptosis was defined by the morphological characteristics detailed by the Committee and by Kerr and co-workers (Kerr et al., 1972), ie, (1) marked condensation of chromatin and cytoplasm; (2) cytoplasmic fragments with or without condensed chromatin; and (3) intra- and extracellular chromatin fragments.
TdT-mediated dUTP nick-end labeling.
Apoptotic cells were detected with the procedure of Sgonc and co-workers (Sgonc et al., 1994) and from morphologic evaluation of H&E-stained tissue. The in situ cell death detection reagent kit (POD) was purchased from Boehringer Mannheim (Indianapolis, IN; Cat. No. 1–684–817). In this method, formalin-fixed, paraffin-embedded liver sections were used for in situ TdT-mediated dUTP nick-end labeling (TUNEL) of 3'-hydroxy-DNA strand breaks. Briefly, 3'-hydroxy-DNA strand breaks were labeled with fluorescein-tagged nucleotides via terminal deoxynucleotidyl transferase and subsequently exposed to horseradish peroxidase-conjugated antifluorescein antibody. Staining was developed with diaminobenzidine (DAB), and sections were counterstained with methyl green. Between 2000 and 2500 hepatocytes per slide were counted in 12–20 randomly selected fields at 400x under a light microscope (Olympus BX50; Lake Success, NY.), and the percent of stained cells (labeling index) was determined.
Proliferating cell nuclear antigen (PCNA) immunohistochemistry.
PCNA immunohistochemistry was conducted as described by Greenwell and colleagues (Greenwell et al., 1991). Briefly, the liver sections mounted on slides were first blocked with casein and then reacted with monoclonal antibody to PCNA (Dako Corporation, Carpentaria, CA). The antibody was then linked with biotinylated goat anti-mouse IgG antibody (Boehringer Mannheim) and labeled with streptavidin-conjugated peroxidase (Jackson Immunoresearch, West Grove, PA). Color was developed by exposing the peroxidase-labeled streptavidin to DAB, forming a brown reaction product. The sections were then counterstained with Gill's hematoxylin. Each slide contained a section of duodenum as a positive control. G0 cells were blue and did not take the PCNA stain, whereas cells in the active stages of the cell cycle were stained brown.
Statistical analysis.
Results are expressed as mean ± SE of groups of 5–25 rats. Homogeneity of variance was tested using the F-max test. If the variances were homogenous, data were analyzed using a completely randomized, factorial ANOVA. Individual comparisons were made with Tukey's 
test. For data sets with nonhomogenous variances, Kruskal-Wallis nonparametric ANOVA was used; individual comparisons were made with Dunn's Multiple Comparisons test. The criterion for significance was p < 0.05 for all comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acute Hepatotoxicity from AFB1
A preliminary study confirmed that AFB1 given ip to rats resulted in dose-dependent hepatotoxicity as marked by elevations in serum ALT and AST activities and histologic changes. A dose of 4 mg AFB1/kg resulted in histologic changes characterized by pronounced periportal lesions accompanied by hemorrhage that appeared to radiate into the midzonal regions in some rats (Fig. 1
). Many parenchymal cells were swollen and anuclear or had pyknotic nuclei characteristic of oncotic necrosis. The portal interstitium was separated from parenchyma, suggesting portal edema, and biliary epithelial cells appeared to be swollen and undergoing oncotic necrosis. Debris was evident in ductal lumens. Also, there appeared to be fewer bile ducts. By contrast, a dose of 1 mg AFB1/kg resulted in no elevation in serum ALT or AST and little or no histologic change. Accordingly, this dose was chosen for further cotreatment studies.
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LPS-Induced Liver Injury
In a preliminary study, LPS, given iv to rats, resulted in dose-dependent hepatotoxicity as marked by elevations in serum ALT and AST activities and histologic changes. A dose of 0.5 x 108 EU LPS/kg produced well-defined patches of midzonal, coagulative hepatocellular oncosis with pronounced neutrophil accumulation in and around the lesions (Fig. 2
). Occasional cells with apoptotic morphology were also present. The coagulative necrosis was associated with changes in markers of liver injury such as elevated serum ALT and AST activities, consistent with previous reports (Hewett et al., 1992; Pearson et al., 1995). By contrast, a dose of 7.4 x 106 EU LPS/kg was associated with little or no hepatocellular oncosis and no increase in serum markers of liver injury. This dose of LPS was chosen for AFB1/LPS cotreatment studies because it produced a modest inflammatory response (i.e., neutrophil influx; see below) without overt hepatotoxicity.
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Development of Hepatic Parenchymal Cell Injury after AFB1/LPS Cotreatment
Based on the preliminary studies described above, rats were treated with either 1 mg AFB1/kg or its vehicle and 4 h later were given 7.4 x 106 EU LPS/kg or its vehicle. Measuring serum ALT and AST activities at various times after the injection of AFB1 assessed hepatic parenchymal cell injury. Serum ALT activity was small at all times examined after treatment of rats either with vehicle, LPS or AFB1 (Fig. 3A
). In contrast, in serum of animals cotreated with AFB1 and LPS there was a trend toward an elevation of serum ALT activity by 12 h that became pronounced and statistically significant by 24 h. Between 48 and 72 h, the activity returned to normal. Similar results were observed for serum AST activity (Fig. 3B).
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These findings were supported by histological examination (Fig. 4
). Histologic alterations were assessed by examining liver sections under a light microscope for necrotic or swollen hepatocytes. At 24 h, most of the livers from the AFB1/Veh-treated group (Fig. 4C) appeared similar to those of the Veh/Veh-treated group (Fig. 4A). However, approximately a quarter of them had occasional, single-cell oncosis in periportal areas. This was seen in all of the rats in this group by 48 h. Infrequent parenchymal cells with disintegrating nuclei were also seen. Most of the livers from Veh/LPS-treated animals were normal (Fig. 4B). In a fourth of the livers from this group, there was an occasional small focus of midzonal oncosis by 24 h. Furthermore, LPS administration resulted in increased numbers of sinusoidal neutrophils as early as 6 h.
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In the AFB1/LPS cotreated group, an increase in neutrophils was evident as early as 6 h. AFB1/LPS cotreatment resulted in swollen cells with oncotic morphology in periportal regions by 12 h. By 24 h, there were widespread areas of single-cell or multiple-cell oncotic necrosis (Fig. 4D
). These findings were most frequent in the periportal areas, but they applied to a lesser extent to midzonal regions. Also there were occasional cell fragments and cells with condensed nuclear chromatin or chromatin fragmentation suggestive of apoptosis. Congestion and hemorrhage in and around the areas of necrosis contributed to a loss of normal sinusoidal architecture within lesioned areas. Thus, the distribution of these lesions was similar to that in animals given AFB1 alone, but their frequency and severity were much more pronounced. No alterations were observed in the centrilobular regions at these times. By 48 h, areas containing necrotic cells had extended to some centrilobular regions. By 72 h, the lesions were resolved and the hepatic architecture restored.
Development of Biliary Injury in AFB1/LPS-Cotreated Rats
Cholangiodestructive cholestasis was estimated through examination of biochemical markers in serum and by histology. Increases in 5'-ND and GGT activities or in bile acid concentration in the serum were not observed in rats treated either with AFB1 or with LPS. In contrast, cotreatment with AFB1 and LPS resulted in pronounced elevation of serum activities of 5'-ND and GGT and bile acids concentration by 12 h (Fig. 5). The 5'-ND activity returned to normal by 48 h, whereas the elevations in GGT activity and bile acids concentration remained elevated until 72 h. AFB1 given alone resulted in an increase in serum ALP at 48 and 72 h (Fig. 5D). In contrast, a more pronounced increase in ALP was observed in cotreated groups at 24 and 48 h.
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At the doses used, treatment with either LPS or AFB1 alone did not result in changes in portal regions compared to vehicle controls, with the exception of increased numbers of small bile ducts in livers from rats treated with AFB1 72 h earlier. In contrast, the cotreated groups had portal edema, swollen and oncotic biliary epithelia, and debris in ductal lumens by 12 h. These changes were more pronounced at 24 h (Fig. 4D
). Furthermore, there appeared to be a decrease in the number of bile ducts at 24 h. By 72 h, there remained little biliary epithelial cell oncosis, and occasionally bile duct epithelial cell hyperplasia appeared to be occurring, as evidenced by layered bile-duct epithelial cells and the appearance of small bile ducts in numbers greater than AFB1 controls. To confirm that hyperplasia had occurred in bile ducts, PCNA immunohistochemistry was conducted. This assay was chosen because it allows identification of all cells that are in the active stages of the cell cycle (i.e., not in G0). AFB1, given alone, resulted in an increase in bile duct epithelial cells stained for PCNA. This effect was enhanced with the co-administration of LPS. LPS, given alone, resulted in an increase in PCNA staining in both hepatic parenchymal and sinusoidal cells. The hepatic parenchymal cells that were stained for PCNA were located primarily in the periportal and midzonal regions but not in the centrilobular region, whereas the sinusoidal cells that were stained for PCNA were panlobular.
Apoptosis
The observation in H&E sections of occasional cytoplasmic cell fragments and cells with condensed nuclear chromatin or chromatin fragmentation suggestive of apoptosis prompted us to evaluate TUNEL staining. None of the treatments resulted in an increase in TUNEL staining at 6 h. At 12 h, LPS treatment resulted in a small yet significant increase in TUNEL staining of cells with apoptotic morphology. This increase was unaffected by cotreatment with AFB1 (Fig. 6). The staining appeared to be associated with single, hepatic parenchymal cells scattered throughout the lobule.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with LPS significantly increased hepatic parenchymal cell necrosis in rats exposed to AFB1: large increases in serum ALT and AST activities were detected at 24 and 48 h after AFB1 exposure. In contrast to this result with AFB1/LPS cotreatment, the same doses of AFB1 or LPS given alone did not increase serum enzyme activities. The changes in aminotransferase activities were consistent with histological evaluation that revealed enhanced hepatocellular oncosis in cotreated animals. Based on the nature of the histopathologic lesions, it appeared that LPS enhanced the toxic effects of AFB1. Hepatic lesions caused by administration of large, hepatotoxic doses of LPS are confined to midzonal areas of liver lobules (Hewett et al., 1992
; Jaeschke et al., 1991; Pearson et al., 1995) (Fig. 2). By contrast, injury from large doses of AFB1 is periportal (Kalengayi and Desmet, 1975; Pestka and Casale, 1990) (Fig. 1). The lesions observed in livers of rats treated with small doses of AFB1 and LPS in these studies originated in the periportal region, a histological picture resembling that of AFB1 rather than LPS administration.
Serum markers indicative of cholestasis (i.e., ALP, GGT and 5'-ND activities, and bile acid concentration) (Zimmerman, 1968) were increased by AFB1/LPS cotreatment. This further suggests that LPS enhanced AFB1-induced toxicity, since such cholestatic changes are a hallmark of AFB1-induced toxicity (Eaton and Heinonen, 1997; Groopman, 1994). Increases in 5'-ND activity and bile acids concentration were seen beginning at 12 h. 5'-ND decreased to normal by 48 h. This correlated with the histologic evaluation, which revealed cessation of bile duct epithelial cell necrosis by 72 h and proliferation of bile duct epithelial cells, another characteristic of aflatoxicosis (Eaton and Heinonen, 1997; Groopman, 1994).
The concentration of bile acids in the serum did not decrease to normal until 72 h. This could be due to a requirement for the portal ducts to be restored before clearance of bile acids could occur. The activity of ALP in the serum also did not decrease to normal until 72 h. This may be due to the release of ALP into the circulation associated with ductal cell hyperplasia (Kaplan, 1986; Seetharam et al., 1986). A small increase in serum ALP was also seen with AFB1 treatment alone at 48 and 72 h, suggesting a modest degree of ductal cell hyperplasia after the administration of AFB1 alone. This was confirmed with PCNA immunohistochemistry, which demonstrated that bile duct epithelial cell hyperplasia occurred after AFB1 administration and that there was a greater degree of hyperplasia in the rats cotreated with LPS.
Although the small dose of LPS did not cause substantial oncosis in this study, it did result in a modest increase in apoptotic cell death, as identified through morphology and TUNEL. It is possible that apoptosis results from small doses of LPS, whereas larger doses cause both oncotic cell death and apoptosis. This scenario has been described with another hepatotoxicant, thioacetamide (Mangipudy et al., 1998). Unlike the effect on oncotic cell death, cotreatment with AFB1 did not enhance apoptosis over that caused by LPS alone. The pathophysiological significance of the small, LPS-induced increase in apoptosis as it relates to acute liver injury is unknown.
It was of interest that neutrophils appeared early, i.e., prior to the onset of injury, in the sinusoids of livers from rats cotreated with AFB1/LPS. An increase in sinusoidal neutrophils was also noticed after a small dose of LPS was given alone. Increases in circulating endotoxin trigger a systemic inflammatory response in a variety of clinical conditions (Bone, 1992; Deitch, 1992; Kelly et al., 1997). In animal models, neutrophils have been implicated as contributors to tissue damage. These include liver injuries induced by LPS and by hepatic ischemia/reperfusion (Jaeschke et al., 1990), as well as by the cholangiolitic hepatotoxicant ANIT (Dahm et al., 1991; Hewett et al., 1992). A hallmark of ANIT hepatotoxicity is an early and marked infiltration of neutrophils (Goldfarb et al., 1962; McLean and Rees, 1958) that precedes cellular injury (Dahm and Roth, 1991; Hewett et al., 1992). Prior neutrophil depletion protects against ANIT hepatotoxicity, suggesting a causal role for these cells in the pathogenesis (Dahm et al., 1991). Like AFB1, ANIT administration to rats results in periportal lesions characterized by injury to parenchymal as well as bile duct epithelial cells. Moreover, plasma LPS is enhanced after ANIT administration, presumably from increased translocation into the blood from the GI tract (Calcamuggi et al., 1992). Accordingly, it is tempting to speculate that the hepatic lesions caused by cotreatment with AFB1 and LPS have a common pathogenesis with those caused by ANIT administration, including perhaps involvement of neutrophils. Further studies will be required to test this hypothesis.
The dosing regimen used in this study (i.e., a barely sub-threshold dose of AFB1, followed 4 h later by a nontoxic dose of LPS) was chosen because, in preliminary studies, it was shown to cause pronounced liver injury. Additional studies will be needed to understand the effects of other doses and different temporal relationships between AFB1 and LPS exposures. With another hepatotoxin, monocrotaline, decreasing the time between exposure to it and LPS markedly enhanced the lethality of the combination (Yee et al., 1998). Substantially increasing the time between administration of AFB1 and LPS would be expected to produce less or no effect, since the effect of each of these agents is reversible at the dose employed. Small doses of LPS given 24–48 h before toxic doses of certain chemicals result in tolerance (Bautista and Spitzer, 1996; Gordon and Rowsey, 1998; Kawabata et al., 1998). Accordingly, it would not be surprising to learn that LPS could enhance, be without effect, or diminish sensitivity to AFB1, depending on the temporal relationship between exposures. This may have importance in considering LPS exposure or underlying inflammation as a determinant of chemical sensitivity and deserves further examination.
There have been several reported incidents of acute aflatoxicosis resulting in death in humans (Krishnamachari et al., 1975; Ngindu et al., 1982; Serck-Hanssen, 1970). One outbreak affecting 17 people resulted in 77% mortality (Chao et al., 1991). The acute lethal dose of AFB1 in humans has been estimated to be approximately 3 mg/kg (Hsieh et al., 1977), a value similar to that of rats (Eaton and Heinonen, 1997; Heathcote and Hibbert, 1978; Newberne and Butler, 1969). Moreover, similar pathologic findings have been reported in humans and rats (Chao et al., 1991; Krishnamachari et al., 1975; Ngindu et al., 1982; Serck-Hanssen, 1970). Given these species similarities, results of the present study in rats raise the possibility that underlying inflammation may in part determine the severity of response in human cases of acute aflatoxicosis.
It has been established by several epidemiological studies in the People's Republic of China that people with hepatitis have a greater risk of developing hepatocellular carcinomas from dietary AFB1 (Groopman et al., 1993; Jacobson et al., 1997; Qian et al., 1994; Ross et al., 1992). A defining feature of hepatitis is an inflammatory response in the liver, and it may be that such inflammation predisposes individuals to the carcinogenic effects of AFB1. Indeed, our results suggest that inflammation is accompanied by hepatic parenchymal cell hyperplasia; such hyperplasia might contribute epigenetically to AFB1-induced carcinogenesis by promoting tumor formation.
In conclusion, when a group of individuals is exposed to a chemical, only a fraction typically experiences injury. This has raised the question as to why certain people are more susceptible to injury. Many determinants of sensitivity exist, genetic polymorphisms in xenobiotic metabolizing enzymes being the most extensively studied. Another possible determinant is increased exposure to inflammatory agents such as LPS. As noted above, clinical and experimental evidence indicates that LPS is present normally in the blood due to GI translocation and that enhanced blood LPS concentration occurs in a wide variety of conditions (Jacob et al., 1977; Nolan, 1981). The results of this study demonstrating augmentation by LPS of the hepatotoxic effects of AFB1 raise the possibility that people and animals may be more sensitive to aflatoxicosis during episodes of modest endotoxemia or other precipitators of a mild inflammatory response.
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ACKNOWLEDGMENTS |
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The authors thank Anya King and Kate Shores for technical assistance. This research was supported by NIH grant ES04139. C.C.B., S.B.Y., and D.A.H. received support from NIH training grant T32 ES07255.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (517) 432-2310. E-mail: rothr{at}msu.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arkesteijn, C. L. (1976). A kinetic method for serum 5'-nucleotidase using stabilized glutamate dehydrogenase. J. Clin. Chem. Clin. Biochem. 14, 155–158.[Web of Science][Medline]
Arthur, M. J., Bentley, I. S., Tanner, A. R., Saunders, P. K., Millward-Sadler, G. H., and Wright, R. (1985). Oxygen-derived free radicals promote hepatic injury in the rat. Gastroenterology 89, 1114–1122.[Web of Science][Medline]
Arthur, M. J., Kowalski-Saunders, P., and Wright, R. (1986). Corynebacterium parvum-elicited hepatic macrophages demonstrate enhanced respiratory burst activity compared with resident Kupffer cells in the rat. Gastroenterology 91, 174–181.[Web of Science][Medline]
Bautista, A. P., and Spitzer, J. J. (1996). Cross-tolerance between acute alcohol intoxication and endotoxemia. Alcohol Clin. Exp. Res. 20, 1395–1400.[Web of Science][Medline]
Bone, R. C. (1992). Toward an epidemiology and natural history of SIRS (systemic inflammatory response syndrome). JAMA 268, 3452–3455.
Bowers, G. N., Jr., and McComb, R. B.(1966). A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin. Chem. 12, 70–89.[Abstract]
Calcamuggi, G., Lanzio, M., Dughera, L., Babini, G., and Emanuelli, G. (1992). Endotoxin tolerance and polymyxin B modify liver damage and cholestasis induced by a single dose of alpha-naphthylisothiocyanate in the rat. Arch. Toxicol. 66, 126–130.[Web of Science][Medline]
Chao, T. C., Maxwell, S. M., and Wong, S. Y. (1991). An outbreak of aflatoxicosis and boric acid poisoning in Malaysia: a clinicopathological study. J. Pathol. 164, 225–233.[Web of Science][Medline]
Dahm, L. J., and Roth, R. A. (1991). Protection against -naphthylisothiocyanate-induced liver injury by decreased hepatic non-protein sulfhydryl content. Biochem. Pharmacol. 42, 1181–1188.[Web of Science][Medline]
Dahm, L. J., Schultze, A. E., and Roth, R. A. (1991). An antibody to neutrophils attenuates alpha-naphthylisothiocyanate-induced liver injury. J. Pharmacol. Exp. Ther. 256, 412–420.
Deitch, E. A. (1992). Multiple organ failure. Pathophysiology and potential future therapy. Ann. Surg. 216, 117–134.[Web of Science][Medline]
Eaton, D. L., and Heinonen, J. T. (1997). Comprehensive Toxicology (I. G. Sipes, C. A. McQueen, and A. J. Gandolfi, Eds.), p. 407. Elsevier Sciences, Oxford, England.
Ghosh, S., Latimer, R. D., Gray, B. M., Harwood, R. J., and Oduro, A. (1993). Endotoxin-induced organ injury. Crit. Care Med. 21, S19–S24.[Web of Science][Medline]
Goldfarb, S., Singer, E. J., and Popper, H. (1962). Experimental cholangitis due to alpha-naphthylisothiocyanate (ANIT). Amer. J. Pathol. 40, 685–698.
Gordon, C. J., and Rowsey, P. J. (1998). Delayed febrile effects of chlorpyrifos: is there cross-tolerance to bacterial lipopolysaccharide? Toxicology 130, 17–28.[Web of Science][Medline]
Greenwell, A., Foley, J. F., and Maronpot, R. R. (1991). An enhancement method for immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissues. Cancer Lett. 59, 251–256.[Web of Science][Medline]
Groopman, J. D. (1994). The Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance. Academic Press, New York.
Groopman, J. D., Wild, C. P., Hasler, J., Junshi, C., Wogan, G. N., and Kensler, T. W. (1993). Molecular epidemiology of aflatoxin exposures: validation of aflatoxin-N7-guanine levels in urine as a biomarker in experimental rat models and humans. Environ. Health Perspect. 99, 107–113.[Web of Science][Medline]
Groopman, J. D., Zhu, J. Q., Donahue, P. R., Pikul, A., Zhang, L. S., Chen, J. S., and Wogan, G. N. (1992). Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi Autonomous Region, People's Republic of China. Cancer Res. 52, 45–52.
Heathcote, J. G., and Hibbert, J. R. (1978). Aflatoxins: Chemical and Biological Aspects. Elsevier Scientific, Amsterdam.
Hewett, J. A., Jean, P. A., Kunkel, S. L., and Roth, R. A. (1993). Relationship between tumor necrosis factor- and neutrophils in endotoxin-induced liver injury. Am. J. Physiol. 265, G1011–G1015.
Hewett, J. A., and Roth, R. A. (1993). Hepatic and extrahepatic pathobiology of bacterial lipopolysaccharides. Pharmacol. Rev. 45, 382–411.[Medline]
Hewett, J. A., and Roth, R. A. (1995). The coagulation system, but not circulating fibrinogen, contributes to liver injury in rats exposed to lipopolysaccharide from gram-negative bacteria. J. Pharmacol. Exp. Ther. 272, 53–62.
Hewett, J. A., Schultze, A. E., Van Cise, S., and Roth, R. A. (1992). Neutrophil depletion protects against liver injury from bacterial endotoxin. Lab. Invest. 66, 347–361.[Web of Science][Medline]
Hsieh, D. P. H., Wong, Z. A., Wong, J. J., Michas, C., and Ruebner, B. H. (1977). Mycotoxins in Human and Animal Health (J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, Eds.), p. 37. Pathotox, Illinois.
Jacob, A. I., Goldberg, P. K., Bloom, N., Degenshein, G. A., and Kozinn, P. J. (1977). Endotoxin and bacteria in portal blood. Gastroenterology 72, 1268–1270.[Web of Science][Medline]
Jacobson, L. P., Zhang, B. C., Zhu, Y. R., Wang, J. B., Wu, Y., Zhang, Q. N., Yu, L. Y., Qian, G.S., Kuang, S. Y., Li, Y. F., Fang, X., Zarba, A., Chen, B., Enger, C., Davidson, N. E., Gorman, M. B., Gordon, G. B., Prochaska, H. J., Egner, P. A., Groopman, J. D., Munoz, A., Helzlsouer, K. J., and Kensler, T. W. (1997). Oltipraz chemoprevention trial in Qidong, People's Republic of China: study design and clinical outcomes. Cancer Epidemiol. Biomarkers Prev. 6, 257–265.[Abstract]
Jaeschke, H., Farhood, A., Bautista, A. P., Spolarics, Z., Spitzer, J. J., and Smith, C. W. (1993). Functional inactivation of neutrophils with a Mac-1 (CD11b/CD18) monoclonal antibody protects against ischemia-reperfusion injury in rat liver. Hepatology 17, 915–923.[Web of Science][Medline]
Jaeschke, H., Farhood, A., and Smith, C. W. (1990). Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J. 4, 3355–3359.[Abstract]
Jaeschke, H., Farhood, A., and Smith, C. W. (1991). Neutrophil-induced liver cell injury in endotoxin shock is a CD11b/CD18-dependent mechanism. Am. J. Physiol. 261, G1051–G1056
Kalengayi, M. M., and Desmet, V. J. (1975). Sequential histological and histochemical study of the rat liver during aflatoxin B1-induced carcinogenesis. Cancer Res. 35, 2845–2852.
Kaplan, M. M. (1986). Serum alkaline phosphatase—another piece is added to the puzzle. Hepatology 6, 526–528.[Web of Science][Medline]
Karmen, A. (1955). A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood. J. Clin. Invest. 34, 131.
Kawabata, A., Hata, T., and Kuroda, R. (1998). Cross tolerance to environmental stress and endotoxin. Life Sci. 62, 327–333.[Web of Science][Medline]
Kelly, J. L., O'Sullivan, C., O'Riordain, M., O'Riordain, D., Lyons, A., Doherty, J., Mannick, J. A., and Rodrick, M. L. (1997). Is circulating endotoxin the trigger for the systemic inflammatory response syndrome seen after injury? Ann. Surg. 225, 530–543.[Web of Science][Medline]
Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257.[Web of Science][Medline]
Krishnamachari, K. A., Bhat, R. V., Nagarajan, V., and Tilak, T. B. (1975). Investigations into an outbreak of hepatitis in parts of western India. Indian J. Med. Res. 63, 1036–1049.[Web of Science][Medline]
Levin, S., Bucci, T. J., Cohen, S. M., Fix, A. S., Hardisty, J. F., LeGrand, E. K., Maronpot, R. R., and Trump, B. F. (1999). The nomenclature of cell death: recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists. Toxicol. Pathol. 27, 484–490.
Lind, R. C., Gandolfi, A. J., Sipes, I. G., and Brown, B. R., Jr. (1984). The involvement of endotoxin in halothane-associated liver injury. Anesthesiology 61, 544–550.[Web of Science][Medline]
Mangipudy, R. S., Rao, P. S., Andrews, A., Bucci, T. J., Witzmann, F. A., and Mehendale, H. M. (1998). Dose-dependent modulation of cell death: apoptosis versus necrosis in thioacetamide hepatotoxicity. Int. J. Toxicol. 17, 193–211.
Mashige, F., Tanaka, N., Maki, A., Kamei, S., and Yamanaka, M. (1981). Direct spectrophotometry of total bile acids in serum. Clin. Chem. 27, 1352–1356.
McLean, M. R., and Rees, K. R. (1958). Hyperplasia of bile ducts induced by alpha-naphthylisothiocyanate: Experimental biliary cirrhosis free from biliary obstruction. J. Pathol. Bacteriol. 76, 175–188.[Web of Science][Medline]
Molvig, J., Baek, L., Christensen, P., Manogue, K. R., Vlassara, H., Platz, P., Nielsen, L. S., Svejgaard, A., and Nerup, J. (1988). Endotoxin-stimulated human monocyte secretion of interleukin 1, tumour necrosis factor alpha, and prostaglandin E2 shows stable interindividual differences. Scand. J. Immunol. 27, 705–716.[Web of Science][Medline]
Moulin, F., Pearson, J. M., Schultze, A. E., Scott, M. A., Schwartz, K. A., Davis, J. M., Ganey, P. E., and Roth, R. A. (1996). Thrombin is a distal mediator of lipopolysaccharide-induced liver injury in the rat. J. Surg. Res. 65, 149–158.[Web of Science][Medline]
Newberne, P. M., and Butler, W. H. (1969). Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: a review. Cancer Res. 29, 236–250.
Ngindu, A., Johnson, B. K., Kenya, P. R., Ngira, J. A., Ocheng, D. M., Nandwa, H., Omondi, T. N., Jansen, A. J., Ngare, W., Kaviti, J. N., Gatei, D., and Siongok, T. A. (1982). Outbreak of acute hepatitis caused by aflatoxin poisoning in Kenya. Lancet 1, 1346–1348.[Web of Science][Medline]
Nolan, J. P. (1975). The role of endotoxin in liver injury. Gastroenterology 69, 1346–1356.[Web of Science][Medline]
Nolan, J. P. (1981). Endotoxin, reticuloendothelial function, and liver injury. Hepatology 1, 458–465.[Web of Science][Medline]
Nolan, J. P. (1989). Intestinal endotoxins as mediators of hepatic injury-an idea whose time has come again. Hepatology 10, 887–891.[Web of Science][Medline]
Pearson, J. M., Schultze, A. E., Jean, P. A., and Roth, R. A. (1995). Platelet participation in liver injury from gram-negative bacterial lipopolysaccharide in the rat. Shock 4, 178–186.[Web of Science][Medline]
Pearson, J. M., Schultze, A. E., Schwartz, K. A., Scott, M. A., Davis, J. M., and Roth, R. A. (1996). The thrombin inhibitor, hirudin, attenuates lipopolysaccharide-induced liver injury in the rat. J. Pharmacol. Exp. Ther. 278, 378–383.
Pestka, J. J., and Casale, W. L. (1990). Food Contamination from Environmental Sources (M. S. Simmons and J. Nriagu, Eds.), pp. 613–638. J. Wiley and Sons, London.
Qian, G. S., Ross, R. K., Yu, M. C., Yuan, J. M., Gao, Y. T., Henderson, B. E., Wogan, G. N., and Groopman, J. D. (1994). A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's Republic of China. Cancer Epidemiol. Biomarkers. Prev. 3, 3–10.[Abstract]
Ross, R. K., Yuan, J. M., Yu, M. C., Wogan, G. N., Qian, G. S., Tu, J.T., Groopman, J. D., Gao, Y. T., and Henderson, B. E. (1992). Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 339, 943–946.[Web of Science][Medline]
Roth, R. A., Harkema, J. R., Pestka, J. P., and Ganey, P. E. (1997). Is exposure to bacterial endotoxin a determinant of susceptibility to intoxication from xenobiotic agents? Toxicol. Appl. Pharmacol. 147, 300–311.[Web of Science][Medline]
Seetharam, S., Sussman, N. L., Komoda, T., and Alpers, D. H. (1986). The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat. Hepatology 6, 374–380.[Web of Science][Medline]
Serck-Hanssen, A. (1970). Aflatoxin-induced fatal hepatitis? A case report from Uganda. Arch. Environ. Health 20, 729–731.[Web of Science][Medline]
Sgonc, R., Boeck, G., Dietrich, H., Gruber, J., Recheis, H., and Wick, G. (1994). Simultaneous determination of cell-surface antigens and apoptosis. Trends Genet. 10, 41–42.[Web of Science][Medline]
Sneed, R. A., Grimes, S. D., Schultze, A. E., Brown, A. P., and Ganey, P. E. (1997). Bacterial endotoxin enhances the hepatotoxicity of allyl alcohol. Toxicol. Appl. Pharmacol. 144, 77–87.[Web of Science][Medline]
Szasz, G. (1974). New substrates for measuring gamma-glutamyl transpeptidase activity. Clin. Chem. Clin. Biochem. 12, 228.
Taylor, M. J., Lafarge-Frayssinet, C., Luster, M. I., and Frayssinet, C. (1991). Increased endotoxin sensitivity following T-2 toxin treatment is associated with increased absorption of endotoxin. Toxicol. Appl. Pharmacol. 109, 51–59.[Web of Science][Medline]
Wood, G. E. (1989). Aflatoxins in domestic and imported foods and feeds. J. Assoc. Off. Anal. Chem. 72, 543–548.[Medline]
Wroblewski, F., and LaDue, J. S. (1956). Serum glutamic-pyruvic transaminase in cardiac hepatic disease. Proc. Soc. Exp. Biol. Med. 91, 569.
Yee, S. B., Ganey, P. E., and Roth, R. A. (1998). Temporal relationships in the augmentation of monocrotoline hepatotoxicity by bacterial endotoxin. Toxicological Sciences 42(suppl.), 366.
Zimmerman, H. J. (1968). The differential diagnosis of jaundice. Med. Clin. North Am. 52, 1417–1444.[Web of Science][Medline]
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