Toxic Effects of Fumonisin in Mouse Liver Are Independent of the Perox
http://www.100md.com
《毒物学科学杂志》
Toxicology & Mycotoxin Research Unit, USDA-Agricultural Research Service, Athens, Georgia 30604–5677
Inorganic Carcinogenesis, LCC, NCI at NIEHS, Research Triangle Park, North Carolina 27709
GlaxoSmithKline, Research Triangle Park, North Carolina 27709
CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
Department of Chemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6
ToxicoGenomics, Chapel Hill, North Carolina 27514
ABSTRACT
Fumonisin mycotoxins occur worldwide in corn and corn-based foods. Fumonisin B1 (FB1) is a rodent liver carcinogen and suspected human carcinogen. It inhibits ceramide synthase and increases tissue sphinganine (Sa) and sphingosine (So) concentrations. Events linking disruption of sphingolipid metabolism and fumonisin toxicity are not fully understood; however, Sa and So were shown to bind mouse recombinant peroxisome proliferator-activated receptor (PPAR) in vitro. To investigate the role of PPAR in fumonisin hepatotoxicity in vivo, wild-type (WT) and PPAR-null mice were fed control diets or diets containing 300 ppm FB1, Fusarium verticillioides culture material (CM) providing 300 ppm FB1, or 500 ppm of the peroxisome proliferator WY-14,643 (WY) for 1 week. WY-fed WT mice exhibited hepatomegaly, an effect not found in WY-fed PPAR-null mice, and WY did not change liver sphingoid base concentrations in either strain. Hepatotoxicity found in FB1- and CM-fed WT and PPAR-null mice was similar, qualitatively different from that found in WY-treated animals, and characterized by increased Sa concentration, apoptosis, and cell proliferation. Transcript profiling using oligonucleotide arrays showed that CM and FB1 elicited similar expression patterns of genes involved in cell proliferation, signal transduction, and glutathione metabolism that were different from that altered by WY. Real-time RT-PCR analysis of gene expression demonstrated PPAR-dependence of lipid metabolism gene expression in WY-treated mice, whereas PPAR-independent alterations of genes in lipid metabolism, and other categories, were found in CM- and FB1-fed mice. Together, these findings demonstrate that FB1- and CM-induced hepatotoxicity in mice does not require PPAR.
Key Words: fumonisin; peroxisome proliferator-activated receptor ; hepatotoxicity.
INTRODUCTION
Fumonisins (Gelderblom et al., 1988) are produced principally by Fusarium verticillioides and F. proliferatum and occur in corn and corn-based foods worldwide (reviewed in depth by the International Programme on Chemical Safety, 2000). Their human health effects are unclear, but a correlation between F. verticillioides or fumonisin contamination of corn and high rates of esophageal cancer in regions of southern Africa and elsewhere has long been recognized (Gelderblom et al., 1991; International Programme on Chemical Safety, 2000). There is also evidence suggesting that fumonisins contribute to liver cancer (Ueno et al., 1997), cardiovascular disease (Fincham et al., 1992), disruption of folate transport/metabolism, and possibly neural tube defects (Marasas et al., 2004). Fumonisins cause the animal diseases associated with F. verticillioides exposure such as leukoencephalomalacia in horses (Kellerman et al., 1990), pulmonary edema in swine (Smith et al., 2000), and hepato- and nephrotoxicities in rodents and other species (Gelderblom et al., 1988; Voss et al., 2001). Fumonisin B1 (FB1) is the most prevalent and thoroughly studied homolog and is hepato- and nephrocarcinogenic when fed to rodents, although significant differences in the organ-specific responses between species (rats vs. mice), strains (F344/N/Nctr vs. BD IX rats), and sexes occur (Gelderblom et al., 1991; Howard et al., 2001).
Most evidence indicates that FB1's mechanism of action is epigenetic (Dragan et al., 2001; International Programme on Chemical Safety, 2000). It and other fumonisins inhibit ceramide synthase (Merrill et al., 2001; Riley et al., 2001), disrupt sphingolipid metabolism, and increase tissue levels of the ceramide precursors sphinganine (Sa) and sphingosine (So) and their downstream metabolites such as sphingosine (sphinganine) 1-phosphate. These events disturb synthesis and turnover of complex sphingolipids as well as sphingolipid-mediated signal transduction of critical pathways governing apoptosis and tissue regeneration (Riley et al., 2001). The precise series of events connecting ceramide synthase inhibition to apoptosis (generally the first morphological finding in target organs of fumonisin-fed rodents (Voss et al., 2001)), necrosis, regeneration, and cancer in rodent liver and kidney have not, however, been elucidated. Lipid peroxidation (Abel et al., 1998), disruption of the cell cycle (Bondy et al., 2000; Lemmer et al., 1999; Ramljak et al., 2000), expression of cytokines and signaling molecules involved in apoptosis such as TNF alpha (Bhandari et al., 2002; Sharma et al., 2002, 2003), changes in cell fatty acid composition (Gelderblom et al., 1997), and disruption of lipid raft function (reviewed in Marasas et al., 2004) also have been reported to be consequences of fumonisin exposure and are likely to play a role in eliciting or modulating its toxicity.
Peroxisome proliferators are a diverse group of compounds that includes hypolipidemic agents, phthalate ester plasticizers, and industrial solvents (Corton et al., 2000; Vanden Heuvel, 1999). Many peroxisome proliferators act as agonists of peroxisome proliferator-activated receptor (PPAR), one of three PPAR subtypes (Corton et al., 2000). A heterodimer of ligand-activated PPAR and another nuclear receptor, retinoid-X-receptor (RXR), binds to DNA response elements and regulates the transcription of target genes (reviewed by Corton et al., 2000; Vanden Heuvel, 1999). The ensuing gene expression changes lead to an orchestration of adaptive responses including increased peroxisome proliferation, cell proliferation, and hepatomegaly. Long-term exposure to peroxisome proliferators results in an increased incidence of liver tumors in mice and rats. The activation of acyl-CoA oxidase (ACO) or other fatty acid -oxidation enzymes that typically occurs upon exposure has been used as a biomarker of peroxisome proliferation. PPAR-null mice lack these typical responses after exposure to peroxisome proliferators (Klaunig et al., 2003; Lee et al., 1995; Peters et al., 1997).
There is conflicting evidence as to the possible role of PPAR in fumonisin-induced hepatotoxicity. Two markers of peroxisome proliferator exposure, peroxisomal -oxidation and carnitine acyltransferase, were not altered in rats fed up to 25 ppm FB1 for 2 years (Gelderblom et al., 1996), a finding that suggests hepatotoxicity in rats occurs independently of peroxisome proliferation. In contrast, increased ACO and CYP4A, markers of PPAR activation, were observed in the liver of FB1-treated rats (Martinez-Larraaga et al., 1996). Additionally, Van Veldhoven et al. (2000) found that some sphingoid bases, including Sa and So (= sphingenine), bind to recombinant mouse PPAR in vitro. It is therefore possible that FB1 and F. verticillioides culture material (CM)-induced hepatotoxicity in mice is mediated, at least in part, by PPAR-dependent signaling. To investigate this possibility, experiments were conducted to compare heptatotoxicity, elevation of sphingoid bases, ACO expression, and transcription profiling in PPAR-null mice and their wild-type (WT) counterparts fed FB1, F. verticillioides CM, or the peroxisome proliferator WY-14,643.
MATERIALS AND METHODS
Control and test diets.
Fumonisin B1 (FB1) was isolated from stirred jar fermentations of F. verticillioides isolate NRRL 13616 according to Miller et al. (1994). Purity of the FB1 was assessed at >98% by comparison to the FB1 standard used to determine the purity of the FB1 for the National Toxicology Program Toxicology and Carcinogenicity Studies of FB1 (National Toxicology Program, 2001) using multiple spectroscopic and physical methods. Culture material (CM) of F. verticillioides isolate MRC 826 (a gift from W. F. O. Marasas, Medical Research Council, Tygerburg, South Africa) was also prepared as previously reported (Voss et al., 2003). Test diets containing 300 ppm FB1, the CM (12% w/w, providing 300 ppm FB1 in the diet), or 500 ppm WY-14,643 (WY) (ChemSyn Science Laboratories, Lenexa, KS) were prepared from basal rodent chow (LM485, Harlan Teklad, Madison, WI) using a Patterson Kelley Blender with intensifier bar. Homogeneity of fumonisin distribution in the FB1 (relative standard deviation = 13%) and CM (relative standard deviation = 25%) diets was determined by HPLC analysis of three random samples of each diet for FB1 (Voss et al., 2003).
Animals.
Wild-type (WT) female SV129 mice and their PPAR-null counterparts (Lee et al., 1995) were individually housed in stainless steel wire-mesh cages in a temperature-controlled room having a 12-h light/dark cycle. The appropriate diet and fresh tap water were provided ad libitum, except during presurgical and prenecropsy fasting periods. After a 2-week acclimation, the mice were anaesthetized with IsoFlo (Isoflurane USP, Abbott Laboratories, North Chicago, IL), and osmotic minipumps (Alzet Model 2001, Durect Corporation, Cupertino, CA) were subcutaneously implanted between the scapulae. The minipumps delivered 1 μl/h of 5-bromo-2'-deoxyuridine (Sigma, St. Louis, MO), 16 mg/ml, dissolved in phosphate buffered saline, pH = 7.0, during the 7-day test period. The WT and PPAR-null mice were then randomly assigned to groups of five mice and fed the control (rodent chow only), FB1, CM, or WY diets for 7 days. Body weight (excluding the osmotic pump) and food consumption were measured on days 0, 4, and 7. The mice were then fasted (3 h), euthanized (CO2), and examined by necropsy. The liver was weighed, and multiple specimens were fixed in 10% neutral buffered formalin (NBF) for histological examination; one-half of the NBF-fixed sections were subsequently (after 48 h) post-fixed in 70% ethanol for determination of hepatocyte proliferation. The remaining liver was cut into cubes (ca. 0.5 cm) and stored frozen (–80°C) for sphingoid base analysis, Western analysis of ACO, or evaluation of gene expression. The experiment was conducted according to the guidelines set forth in the Guide for the Care and Use of Laboratory Animals and the USDA-ARS Policies and Procedures for the Humane Animal Care and Use Committee, USDA Agricultural Research Service, Richard B. Russell Agricultural Research Laboratory, Athens, GA.
Determination of level of liver sphingoid bases.
Liver sphinganine (Sa) and sphingosine (So) concentrations were determined using previously described extraction and HPLC methods (Riley et al., 1994).
Western blot.
Liver lysates were prepared in 250 mM sucrose, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA with protease inhibitors (0.2 mM PMSF, 0.1% aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin) as previously described (Fan et al., 2003). Fifty μg of whole-cell lysate was subjected to 12% SDS–PAGE followed by transfer to nitrocellulose membranes. Immunoblots were developed using primary antibodies against acyl-CoA oxidase (ACO) (a gift from Dr. S. Alexson, Huddinge University Hospital, Huddinge, Sweden) or CYP4A (GenTest, Waltham, MA) and appropriate secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of chemiluminescent substrate enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Histopathology and hepatocyte proliferation.
Hematoxylin- and eosin-stained sections (4–6 microns thick) were prepared and microscopically examined. Examinations were done without knowledge of the animals' strain or treatment. Each tissue was scored for the presence of lesions consistent with fumonisin exposure, and the number of apoptotic cells and mitotic figures were counted (Sharma et al., 2002). The procedure was repeated and the results averaged.
Nuclei that incorporated BrdU were identified by immunohistochemistry (Miller et al., 2001). Light microscopy was performed using a Microphot microscope (Nikon, Melville, NY) with a Dage CCD color video camera (DAGE-MTI, Inc., Michigan City, IN). The hepatocytes were analyzed using the Cytology Histology Recognition Identification System (CHRIS, Sverdrup Medical/Life Sciences Imaging Systems, Fort Walton Beach, FL). At least 1000 cells were counted for each animal. Cells that incorporated BrdU were identified by red pigmented nuclei. Ten to fifteen fields were counted. The labeling index (LI) was calculated by dividing the number of labeled hepatocyte nuclei by the total number of hepatocyte nuclei counted, and the results were expressed as a percentage.
RNA isolation and analysis of gene expression using oligonucleotide arrays.
Three mice were analyzed from each of six treatment groups, for a total of 18 analyses. Hepatic RNA was isolated using a modified guanidium isothiocyanate method (TRIzol, InvitrogenTM, Carlsbad, CA) and was further purified using silica membrane spin columns (RNEasy Total RNA Kit, Qiagen, Valencia, CA). RNA integrity was assessed by ethidium bromide staining followed by resolution on denaturing agarose gels and also by the RNA 6000 LabChip Kit using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). For each sample, 15 μg of biotin-labeled cRNA was generated from 10 μg total RNA and hybridized to GeneChip Test3 Arrays (Affymetrix, Inc., Santa Clara, CA) to determine quality. Subsequently, the same samples were hybridized to Murine GeneChipTM U74Av.2 oligonucleotide arrays (Affymetrix). All procedures were carried out according to the manufacturer's recommendations, using the antibody amplification technique.
Images were initially processed using the MAS 5.0 software (Affymetrix). Hybridization quality was assessed by visual inspection of the image and from a report generated by MAS 5.0. Criteria for an acceptable hybridization were as follows: background < 100, noise (RawQ) < 5, 3'/5' ratio for select housekeeping genes < 4. Hybridizations not meeting these criteria were repeated, beginning at the target preparation step. The data were analyzed and statistically filtered using Rosetta Resolver version 3.0 software (Rosetta Inpharmatics, Kirkland, WA). The threshold for significance was set at p 0.001, and genes which exhibited a 1.5-fold or –1.5-fold change were reported as a fold-change relative to the corresponding control. Similarly regulated genes were visualized using CLUSTER and TreeView (Eisen et al., 1998). Genes were grouped into functional classes with the help of KEGG (http://www.kegg.org) and using Gene Ontology (http://www.geneontology.org) identifiers in the U74Av2 template (version accessed Aug. 1, 2003 from http://www.affymetrix.com). Identification of ESTs was facilitated by euGenes (http://eugenes.org/mouse/).
Real-time RT-PCR analysis.
The levels of expression of the selected genes were quantified using real-time RT-PCR analysis. Briefly, total RNA was extracted as described above and reverse transcribed with MuLV reverse transcriptase and oligo-dT primers. The forward and reverse primers for selected genes (Table 1) were designed using Primer Express software, v2.0 (Applied Biosystems, Foster City, CA). The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis. The relative differences in expression between groups were expressed using cycle time (Ct) values as follows: the Ct values of the genes were first normalized with -actin of the same sample, and then the relative differences between control and treatment groups were calculated and expressed as relative increases, setting the control as 100%. Assuming that the Ct value is reflective of the initial starting copy and that there is 100% efficiency, a difference of one cycle is equivalent to a two-fold difference in starting copy.
Statistics.
Statistical procedures generally followed the scheme of Gad and Weil (1989) using SAS (SAS Institute, Cary, NC) or SigmaStat (Jandel Scientific Software, San Rafael, CA) software. FB1-, CM-, and WY-treated and controls of each strain were compared using ANOVA followed by the Duncan's multiple range test to identify differences among groups (parametric, homogenous data), the Kruskal-Wallis test followed by the distribution free multiple comparisons (nonparametric data) or Fischer's least significant differences test (incidence data). Two-group comparisons of WT and PPAR-null groups receiving the same diet were done by Students t-test. Tests were two tailed, and significance was judged at p < 0.05.
RESULTS
Appearance, Body Weight and Food Consumption
With one exception, a CM-fed PPAR-null mouse that became inactive and not responsive to touch on the last day of exposure, the appearance and activity of the animals were unremarkable. FB1- and CM-fed mice of both strains lost weight (Table 2). Cumulative loss averaged from 2.0 (FB1-fed WT) to 6.5% (CM-fed PPAR-null), but compared to their control groups, statistical significance was found only in FB1- and CM-fed PPAR-null mice. Cumulative weight gain of WY-fed WT mice was significantly increased compared to the WT controls. No significant differences in food consumption were found. Mean daily food consumptions (g/kg body weight) for the WT groups were: control = 237 ± 10.5 (SD), WY = 238 ± 21.5, FB1 = 245 ± 33.9, and CM = 228 ± 15.2. Values for the PPAR-null groups were: control = 232 ± 37.1, WY = 233 ± 26.9, FB1 = 212 ± 31.3, CM = 211 ± 27.2.
Necropsy and Liver Weights
No noteworthy gross lesions were found at necropsy. Final (fasted) body weights of the PPAR-null FB1- and CM-fed groups were significantly reduced (9 to 10%) compared to their controls (Table 3), while no significant differences in final body weight were found among the four WT groups. Both FB1 and the CM significantly increased the absolute (32 to 35%) and relative liver weights (37 to 43%) of WT mice (Table 3). Liver weights of PPAR-null mice fed FB1 or the CM were slightly higher than those of their controls; statistical significance was demonstrated only for the relative liver weight of the CM-fed group (28% greater than the controls). WY significantly increased the absolute and relative liver weights of the WT mice but had no significant effect on liver weights of the PPAR-null animals, as expected.
Microscopic Pathology and Hepatocyte Proliferation
Microscopic appearance of the livers from WT and PPAR control mice was unremarkable. The only observation of note was a mild degree of hepatocellular vacuolation in one WT and all PPAR-null animals. The vacuoles' appearance was consistent with lipid deposition but was not confirmed histochemically. Two to four mice from each of the remaining treatment/strain combinations showed a similar pattern of hepatocellular vacuolation. WY caused marked hepatocytomegaly and cytoplasmic eosinophilia in the WT mice. Densely staining chromatin and obviously increased numbers of mitotic figures were also noted. That WY had no significant effect on the microscopic appearance of livers from PPAR-null mice is consistent with previous observations (Lee et al., 1995; Peters et al., 1997).
FB1 and the CM caused liver lesions in both strains that were consistent with those previously found in fumonisin-exposed mice (Sharma et al., 2002). The lesions included focal apoptosis, mitotic figures, focal oncotic or coagulative necrosis (3–4 mice/group), focal or generalized variation in cell (anisocytosis) and nucleus (anisokaryosis) size (3–5 mice/group), focal acute inflammation (2–3 FB1-treated mice/group), or a slight amount of focal bile duct hyperplasia (2 FB1-treated and 2 CM-treated PPAR-null mice). Overall group mean pathology scores were 1.5 to 1.6 (Table 4), indicating that FB1 and the CM caused a similar degree of liver injury in both strains. The average number of hepatic apoptotic foci per mouse induced by FB1 or CM tended to be slightly higher, and liver mitotic figure counts tended to be slightly lower in the PPAR-null mice (Table 4), but no statistically significant differences for either variable were found between the two strains.
WT mice exhibited significant increases in cell proliferation after exposure to WY, CM, or FB1 as determined using BrdU positive nuclei as the experimental endpoint (Fig. 1). The findings corroborated the histopathology results indicating that cell proliferation was approximately equal in the CM- and FB1-fed WT groups. While PPAR-null mice did not exhibit cell proliferation after WY exposure, cell proliferation rates of FB1- and CM-fed PPAR-null mice did not differ from those of their corresponding WT groups. Thus, histopathology and BrdU results indicate that apoptosis and cell proliferation stimulated by FB1 and CM were PPAR-independent.
Liver Sphingoid Base Concentrations and ACO Protein Expression
Both FB1 and CM increased hepatic Sa concentrations (Fig. 2a) and Sa/So ratios (Fig. 2b) in the WT and PPAR-null strains; So concentrations were unaffected by both. Liver Sa concentrations in the PPAR-null mice fed FB1 were significantly less than those of their WT counterparts. They were also slightly lower in CM-fed PPAR-null than in CM-fed WT mice, but the difference was not statistically significant. However, Sa/So ratios of the groups fed FB1 or CM were not significantly different from one another (8.7 ± 3.1 (SD) for FB1-fed PPAR-null and 12.3 ± 2.6 for FB1-fed WT). WY had no effect on hepatic sphingoid base concentrations in either strain. WY exposure increased the levels of liver ACO and CYP4A in WT but not PPAR-null mice, whereas FB1 and CM did not cause any changes in the expression of these markers of peroxisome proliferation in either strain (data not shown).
Transcriptional Programs Regulated by Fumonisin
We identified 1459 genes that were significantly different between two or more groups (p 0.001; 1.5-fold or –1.5-fold change) as outlined in the Materials and Methods. Most of these genes were altered by WY (1217 genes), consisting of 732 up-regulated and 485 down-regulated genes. FB1 up-regulated 180 genes and down-regulated 193. CM exhibited a similar response, up-regulating 200 genes and down-regulating 203. Many of the genes unique to WY (a total of 1012 genes) have been discussed in previous studies (Anderson et al., 2004a,b; Corton et al., 2004) and will not be discussed here. We focus here on the genes regulated by CM and FB1, including those genes that are also altered by WY exposure.
The genes were classified into major groups based on their expression behavior (Fig. 3). The first two classes were those regulated by CM (56 genes) or FB1 (48 genes) only. The third and fourth classes were genes regulated by CM and WY or FB1 and WY. There were a total of 49 and 27 genes in these classes, respectively. Most of the genes in these classes exhibited similar expression by CM and WY or FB1 and WY. In both classes 33% of the total number of genes exhibited opposite expression behavior. The fifth class of genes consisted of those regulated by both CM and FB1 but not WY. There were 138 genes in this class. The sixth class consisted of genes regulated by all three compounds. There were a total of 160 genes in this category. This class was dominated by genes that exhibited similar regulation by all three treatments (82%) and most of the genes regulated by either CM or FB1 were regulated by both treatments (62%). Furthermore, all of the genes coregulated by CM and FB1 were not only regulated in a similar direction, but the level of up- or down-regulation was similar. These results demonstrate that CM and FB1 exhibit an overlapping set of genes. Furthermore, the majority of genes regulated by CM and FB1 do not overlap with the genes regulated by WY.
The genes regulated by CM or FB1 and WY fell into a number of functional categories including lipid metabolism, cell proliferation, signal transduction, phase I and II xenobiotic metabolism, and glutathione metabolism (Table 5) (Supplementary Table 1 for the complete list). Real-time RT-PCR was used to confirm the expression changes of 27 genes that fell into many of these categories (Table 6). In these experiments we examined the expression changes in WT and PPAR-null mice after compound treatment. In general, the expression changes observed by transcript profiling were corroborated by real-time RT-PCR. However, there were a few exceptions. Fabp1 in WY-treated WT mice exhibited down-regulation by profiling but exhibited up-regulation by real-time RT-PCR, and Cdkn2b in WT mice was down-regulated by all three treatments by profiling, whereas there was up-regulation by CM and FB1 by real-time RT-PCR. CM and FB1 exhibited an identical pattern of changes in WT mice. Almost all of the changes were not only in the same direction but of approximately the same magnitude in WT and PPAR-null mice, demonstrating that PPAR is not required for CM or FB1 to alter gene expression. Exceptions were Cyp4a10, in which CM and FB1 decreased expression in WT but increased expression in PPAR-null mice, and Cdkn2a, which exhibited induction in WT but not PPAR-null mice after FB1 treatment. As expected, WY required PPAR for the majority of changes. PPAR-null mice exhibited some induction of Cyp4a10 and Mte1 after WY exposure. PPAR-independent induction of Cyp4a10 and Cyp4a14 by WY has been observed earlier (Anderson et al., 2004a,b). These findings demonstrate that the gene expression changes after CM or FB1 exposure are PPAR independent and identify novel targets related to FB1 toxicity.
DISCUSSION
There are few studies addressing whether PPAR-dependent signaling pathways are involved in fumonisin toxicity, and the reported results are inconsistent. Gelderblom et al. (1996) concluded that peroxisome proliferation did not play a role in FB1-induced hepatotoxicity in rats, whereas Martinez-Larraaga et al. (1996) observed that FB1 increased CYP4A1 apoprotein levels and -oxidation of palmitoyl coenzyme A and suggested peroxisome proliferation could play a role. FB1 is clearly hepatotoxic (Sharma et al., 2002, 2003) and hepatocarcinogenic (Howard et al., 2001) to female mice; however, its effect on PPAR-dependent pathways in mice is unknown. To address this question, we compared the hepatotoxic effects of FB1 in wild-type (WT) and PPAR-null mice. Fungal culture materials have been used as a fumonisin source in animal feeding studies and have been shown to elicit qualitatively similar morphologic and sphingolipid effects in rodents as FB1 (Voss et al., 2001). However, the fungal culture materials contain other fumonisins and secondary metabolites in addition to FB1 that might exert biological activity (International Programme on Chemical Safety, 2000; Voss et al., 2001). We therefore also compared the hepatotoxic effects of a commonly used F. verticillioides CM in the two mouse stains to determine if the fungus produced any other fumonisins or nonfumonisin compounds having significant PPAR-dependent activity.
The hepatic response of the two strains to WY clearly differed. The absence of liver hypertrophy, hepatocellular cytomegaly, cytoplasmic eosinophilia, mitotic figures, and BrdU positive nuclei in the PPAR-null mice confirmed that PPAR-mediated signaling was disrupted in this strain as seen in earlier studies (summarized in Corton et al., 2000). Contrary to our hypothesis, however, the absence of PPAR-dependent signaling did not significantly abrogate the typical hepatotoxic responses to FB1 or CM. That the expression of ACO, a marker of peroxisome proliferation, and CYP4A was not increased in either the WT or PPAR-null strains is evidence that PPAR-dependent signaling, including induction of peroxisome proliferation, plays no significant role in fumonisin or F. verticillioides hepatotoxicity in mice.
To gain insight into the genes altered by FB1 exposure, we generated transcript profiles from the livers of wild-type mice exposed to CM, FB1, and WY. There was a close correlation in the expression pattern between CM and FB1. Approximately 62% of all genes altered by either CM or FB1 treatments were not only altered in the same direction, but the magnitude of the changes was very similar. In contrast, most of the genes regulated by CM and FB1 do not overlap with those regulated by WY. As both WY and FB1 increase cell proliferation under these conditions, it was not surprising that many of the genes that WY and FB1 have in common are those involved in cell fate, including many involved in the cell cycle. We examined the expression of a number of overlapping genes by RT-PCR in both WT and PPAR-null mice and showed that CM and FB1 alter the expression of genes in a number of different categories independently of PPAR, whereas WY required PPAR for most of the changes. This provides further evidence that FB1 does not act like a typical peroxisome proliferator.
There are only a few reports on fumonisin-induced alteration of gene expression patterns in vivo. Rats fed 250 ppm FB1 for up to 5 weeks exhibited a progressive increase in the expression of several genes involved in cell proliferation and regeneration including hepatocyte growth factor (HGF), transforming growth factor (TGF), and transforming growth factor-1 (TGF-1), as well as the oncongene c-myc (Lemmer et al., 1999). Bondy et al., (2000) found that FB1 increased liver cyclin D1 protein without affecting its mRNA expression in rats treated with FB1 for 6 days. Ramljak et al. (2000) likewise reported a dose-related overexpression of hepatic cyclin D1 in rats fed diets containing up to 250 ppm FB1 for 3 weeks. Overexpression did not result from increased cyclin D1 mRNA transcription but from post-translational stabilization of cyclin D1 through a process dependent on glycogen synthase kinase 3 and Akt. Together, these findings suggested that cell cycle disruption, particularly disruption of G1/S transition, is a critical event in FB1 carcinogenesis. Our results differed from these reports in that cyclin D1 transcription was increased in both mouse strains by fumonisin exposure, whereas the expression of HGF, TGF, TGF-1, or c-myc was not significantly altered. As in rats (Bondy et al., 2000), FB1 and CM increased transcription of the cyclin dependent kinase inhibitor cdkn1A (p21) in mice.
Sharma et al. (2003) found increased expression of mRNA for genes in the livers of mice treated with 2.25 mg/kg body weight FB1 for five consecutive days that are involved in apoptosis (bax, c-myc, caspase 8), cytokine signaling (IL-1 receptor agonist), and tumor necrosis factor (TNF) or Fas signaling (FADD, FAP, FAF, TRAIL, TRADD, RIP, and TNFR1) pathways. In the present study, the expression of two genes involved in apoptosis (clusterin, apoptosis inhibitor 6) was significantly altered by FB1 or CM. The direct and indirect (i.e., mediated by sphingoid base accumulation, decreased ceramide biosynthesis, or complex sphingolipid depletion) effects of fumonisins on gene expression are complex, and determining their mechanistic implications in regard to fumonisin hepatotoxicity or carcinogenicity is beyond the scope of this investigation. However, this list of regulated genes will provide a basis for hypotheses explaining linkages between gene expression changes and phenotypic effects of fumonisins. Future studies should consider dose- and time course-related effects as well as species- and strain-related differences in the expression of gene products. The latter is particularly important when using genetically modified animal models, as demonstrated by the results of Sharma et al. (2003), which showed that FB1 significantly increased the expression of CD95 Ligand (FasL) and other signaling molecules in TNF-null mice compared to their corresponding wild-type controls, thereby offering an plausible explanation for the unexpected increase in severity of hepatotoxicity in FB1-treated TNF-null mice (Sharma et al., 2002).
In summary, results from this in vivo feeding study showed that the hepatotoxic mechanism of FB1 in mice does not involve PPAR-mediated signaling pathways and further indicate that F. verticillioides CM does not contain other metabolites having significant peroxisome proliferator activity. The specific sequence of events linking the inhibition of ceramide synthase by fumonisins to altered gene expression, apoptosis, mitosis, and overt hepatotoxicity in rodents remains to be elucidated.
SUPPLEMENTARY DATA
Supplementary data are available online at www.toxsci.oxfordjournals.org.
NOTES
2 Current address: Bayer Corporation, Research Triangle Park, NC 27709.
3 Current address: Toxicogenomics Program, National Health and Environmental Effects Research Lab, US-EPA, Research Triangle Park, NC 27711.
ACKNOWLEDGMENTS
The authors thank N. Brice, R. de la Campa, P. Malcom, J. Showker, P. Stancel, and E. Wray for their expert assistance. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Disclaimer: Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
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Inorganic Carcinogenesis, LCC, NCI at NIEHS, Research Triangle Park, North Carolina 27709
GlaxoSmithKline, Research Triangle Park, North Carolina 27709
CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
Department of Chemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6
ToxicoGenomics, Chapel Hill, North Carolina 27514
ABSTRACT
Fumonisin mycotoxins occur worldwide in corn and corn-based foods. Fumonisin B1 (FB1) is a rodent liver carcinogen and suspected human carcinogen. It inhibits ceramide synthase and increases tissue sphinganine (Sa) and sphingosine (So) concentrations. Events linking disruption of sphingolipid metabolism and fumonisin toxicity are not fully understood; however, Sa and So were shown to bind mouse recombinant peroxisome proliferator-activated receptor (PPAR) in vitro. To investigate the role of PPAR in fumonisin hepatotoxicity in vivo, wild-type (WT) and PPAR-null mice were fed control diets or diets containing 300 ppm FB1, Fusarium verticillioides culture material (CM) providing 300 ppm FB1, or 500 ppm of the peroxisome proliferator WY-14,643 (WY) for 1 week. WY-fed WT mice exhibited hepatomegaly, an effect not found in WY-fed PPAR-null mice, and WY did not change liver sphingoid base concentrations in either strain. Hepatotoxicity found in FB1- and CM-fed WT and PPAR-null mice was similar, qualitatively different from that found in WY-treated animals, and characterized by increased Sa concentration, apoptosis, and cell proliferation. Transcript profiling using oligonucleotide arrays showed that CM and FB1 elicited similar expression patterns of genes involved in cell proliferation, signal transduction, and glutathione metabolism that were different from that altered by WY. Real-time RT-PCR analysis of gene expression demonstrated PPAR-dependence of lipid metabolism gene expression in WY-treated mice, whereas PPAR-independent alterations of genes in lipid metabolism, and other categories, were found in CM- and FB1-fed mice. Together, these findings demonstrate that FB1- and CM-induced hepatotoxicity in mice does not require PPAR.
Key Words: fumonisin; peroxisome proliferator-activated receptor ; hepatotoxicity.
INTRODUCTION
Fumonisins (Gelderblom et al., 1988) are produced principally by Fusarium verticillioides and F. proliferatum and occur in corn and corn-based foods worldwide (reviewed in depth by the International Programme on Chemical Safety, 2000). Their human health effects are unclear, but a correlation between F. verticillioides or fumonisin contamination of corn and high rates of esophageal cancer in regions of southern Africa and elsewhere has long been recognized (Gelderblom et al., 1991; International Programme on Chemical Safety, 2000). There is also evidence suggesting that fumonisins contribute to liver cancer (Ueno et al., 1997), cardiovascular disease (Fincham et al., 1992), disruption of folate transport/metabolism, and possibly neural tube defects (Marasas et al., 2004). Fumonisins cause the animal diseases associated with F. verticillioides exposure such as leukoencephalomalacia in horses (Kellerman et al., 1990), pulmonary edema in swine (Smith et al., 2000), and hepato- and nephrotoxicities in rodents and other species (Gelderblom et al., 1988; Voss et al., 2001). Fumonisin B1 (FB1) is the most prevalent and thoroughly studied homolog and is hepato- and nephrocarcinogenic when fed to rodents, although significant differences in the organ-specific responses between species (rats vs. mice), strains (F344/N/Nctr vs. BD IX rats), and sexes occur (Gelderblom et al., 1991; Howard et al., 2001).
Most evidence indicates that FB1's mechanism of action is epigenetic (Dragan et al., 2001; International Programme on Chemical Safety, 2000). It and other fumonisins inhibit ceramide synthase (Merrill et al., 2001; Riley et al., 2001), disrupt sphingolipid metabolism, and increase tissue levels of the ceramide precursors sphinganine (Sa) and sphingosine (So) and their downstream metabolites such as sphingosine (sphinganine) 1-phosphate. These events disturb synthesis and turnover of complex sphingolipids as well as sphingolipid-mediated signal transduction of critical pathways governing apoptosis and tissue regeneration (Riley et al., 2001). The precise series of events connecting ceramide synthase inhibition to apoptosis (generally the first morphological finding in target organs of fumonisin-fed rodents (Voss et al., 2001)), necrosis, regeneration, and cancer in rodent liver and kidney have not, however, been elucidated. Lipid peroxidation (Abel et al., 1998), disruption of the cell cycle (Bondy et al., 2000; Lemmer et al., 1999; Ramljak et al., 2000), expression of cytokines and signaling molecules involved in apoptosis such as TNF alpha (Bhandari et al., 2002; Sharma et al., 2002, 2003), changes in cell fatty acid composition (Gelderblom et al., 1997), and disruption of lipid raft function (reviewed in Marasas et al., 2004) also have been reported to be consequences of fumonisin exposure and are likely to play a role in eliciting or modulating its toxicity.
Peroxisome proliferators are a diverse group of compounds that includes hypolipidemic agents, phthalate ester plasticizers, and industrial solvents (Corton et al., 2000; Vanden Heuvel, 1999). Many peroxisome proliferators act as agonists of peroxisome proliferator-activated receptor (PPAR), one of three PPAR subtypes (Corton et al., 2000). A heterodimer of ligand-activated PPAR and another nuclear receptor, retinoid-X-receptor (RXR), binds to DNA response elements and regulates the transcription of target genes (reviewed by Corton et al., 2000; Vanden Heuvel, 1999). The ensuing gene expression changes lead to an orchestration of adaptive responses including increased peroxisome proliferation, cell proliferation, and hepatomegaly. Long-term exposure to peroxisome proliferators results in an increased incidence of liver tumors in mice and rats. The activation of acyl-CoA oxidase (ACO) or other fatty acid -oxidation enzymes that typically occurs upon exposure has been used as a biomarker of peroxisome proliferation. PPAR-null mice lack these typical responses after exposure to peroxisome proliferators (Klaunig et al., 2003; Lee et al., 1995; Peters et al., 1997).
There is conflicting evidence as to the possible role of PPAR in fumonisin-induced hepatotoxicity. Two markers of peroxisome proliferator exposure, peroxisomal -oxidation and carnitine acyltransferase, were not altered in rats fed up to 25 ppm FB1 for 2 years (Gelderblom et al., 1996), a finding that suggests hepatotoxicity in rats occurs independently of peroxisome proliferation. In contrast, increased ACO and CYP4A, markers of PPAR activation, were observed in the liver of FB1-treated rats (Martinez-Larraaga et al., 1996). Additionally, Van Veldhoven et al. (2000) found that some sphingoid bases, including Sa and So (= sphingenine), bind to recombinant mouse PPAR in vitro. It is therefore possible that FB1 and F. verticillioides culture material (CM)-induced hepatotoxicity in mice is mediated, at least in part, by PPAR-dependent signaling. To investigate this possibility, experiments were conducted to compare heptatotoxicity, elevation of sphingoid bases, ACO expression, and transcription profiling in PPAR-null mice and their wild-type (WT) counterparts fed FB1, F. verticillioides CM, or the peroxisome proliferator WY-14,643.
MATERIALS AND METHODS
Control and test diets.
Fumonisin B1 (FB1) was isolated from stirred jar fermentations of F. verticillioides isolate NRRL 13616 according to Miller et al. (1994). Purity of the FB1 was assessed at >98% by comparison to the FB1 standard used to determine the purity of the FB1 for the National Toxicology Program Toxicology and Carcinogenicity Studies of FB1 (National Toxicology Program, 2001) using multiple spectroscopic and physical methods. Culture material (CM) of F. verticillioides isolate MRC 826 (a gift from W. F. O. Marasas, Medical Research Council, Tygerburg, South Africa) was also prepared as previously reported (Voss et al., 2003). Test diets containing 300 ppm FB1, the CM (12% w/w, providing 300 ppm FB1 in the diet), or 500 ppm WY-14,643 (WY) (ChemSyn Science Laboratories, Lenexa, KS) were prepared from basal rodent chow (LM485, Harlan Teklad, Madison, WI) using a Patterson Kelley Blender with intensifier bar. Homogeneity of fumonisin distribution in the FB1 (relative standard deviation = 13%) and CM (relative standard deviation = 25%) diets was determined by HPLC analysis of three random samples of each diet for FB1 (Voss et al., 2003).
Animals.
Wild-type (WT) female SV129 mice and their PPAR-null counterparts (Lee et al., 1995) were individually housed in stainless steel wire-mesh cages in a temperature-controlled room having a 12-h light/dark cycle. The appropriate diet and fresh tap water were provided ad libitum, except during presurgical and prenecropsy fasting periods. After a 2-week acclimation, the mice were anaesthetized with IsoFlo (Isoflurane USP, Abbott Laboratories, North Chicago, IL), and osmotic minipumps (Alzet Model 2001, Durect Corporation, Cupertino, CA) were subcutaneously implanted between the scapulae. The minipumps delivered 1 μl/h of 5-bromo-2'-deoxyuridine (Sigma, St. Louis, MO), 16 mg/ml, dissolved in phosphate buffered saline, pH = 7.0, during the 7-day test period. The WT and PPAR-null mice were then randomly assigned to groups of five mice and fed the control (rodent chow only), FB1, CM, or WY diets for 7 days. Body weight (excluding the osmotic pump) and food consumption were measured on days 0, 4, and 7. The mice were then fasted (3 h), euthanized (CO2), and examined by necropsy. The liver was weighed, and multiple specimens were fixed in 10% neutral buffered formalin (NBF) for histological examination; one-half of the NBF-fixed sections were subsequently (after 48 h) post-fixed in 70% ethanol for determination of hepatocyte proliferation. The remaining liver was cut into cubes (ca. 0.5 cm) and stored frozen (–80°C) for sphingoid base analysis, Western analysis of ACO, or evaluation of gene expression. The experiment was conducted according to the guidelines set forth in the Guide for the Care and Use of Laboratory Animals and the USDA-ARS Policies and Procedures for the Humane Animal Care and Use Committee, USDA Agricultural Research Service, Richard B. Russell Agricultural Research Laboratory, Athens, GA.
Determination of level of liver sphingoid bases.
Liver sphinganine (Sa) and sphingosine (So) concentrations were determined using previously described extraction and HPLC methods (Riley et al., 1994).
Western blot.
Liver lysates were prepared in 250 mM sucrose, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA with protease inhibitors (0.2 mM PMSF, 0.1% aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin) as previously described (Fan et al., 2003). Fifty μg of whole-cell lysate was subjected to 12% SDS–PAGE followed by transfer to nitrocellulose membranes. Immunoblots were developed using primary antibodies against acyl-CoA oxidase (ACO) (a gift from Dr. S. Alexson, Huddinge University Hospital, Huddinge, Sweden) or CYP4A (GenTest, Waltham, MA) and appropriate secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of chemiluminescent substrate enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Histopathology and hepatocyte proliferation.
Hematoxylin- and eosin-stained sections (4–6 microns thick) were prepared and microscopically examined. Examinations were done without knowledge of the animals' strain or treatment. Each tissue was scored for the presence of lesions consistent with fumonisin exposure, and the number of apoptotic cells and mitotic figures were counted (Sharma et al., 2002). The procedure was repeated and the results averaged.
Nuclei that incorporated BrdU were identified by immunohistochemistry (Miller et al., 2001). Light microscopy was performed using a Microphot microscope (Nikon, Melville, NY) with a Dage CCD color video camera (DAGE-MTI, Inc., Michigan City, IN). The hepatocytes were analyzed using the Cytology Histology Recognition Identification System (CHRIS, Sverdrup Medical/Life Sciences Imaging Systems, Fort Walton Beach, FL). At least 1000 cells were counted for each animal. Cells that incorporated BrdU were identified by red pigmented nuclei. Ten to fifteen fields were counted. The labeling index (LI) was calculated by dividing the number of labeled hepatocyte nuclei by the total number of hepatocyte nuclei counted, and the results were expressed as a percentage.
RNA isolation and analysis of gene expression using oligonucleotide arrays.
Three mice were analyzed from each of six treatment groups, for a total of 18 analyses. Hepatic RNA was isolated using a modified guanidium isothiocyanate method (TRIzol, InvitrogenTM, Carlsbad, CA) and was further purified using silica membrane spin columns (RNEasy Total RNA Kit, Qiagen, Valencia, CA). RNA integrity was assessed by ethidium bromide staining followed by resolution on denaturing agarose gels and also by the RNA 6000 LabChip Kit using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). For each sample, 15 μg of biotin-labeled cRNA was generated from 10 μg total RNA and hybridized to GeneChip Test3 Arrays (Affymetrix, Inc., Santa Clara, CA) to determine quality. Subsequently, the same samples were hybridized to Murine GeneChipTM U74Av.2 oligonucleotide arrays (Affymetrix). All procedures were carried out according to the manufacturer's recommendations, using the antibody amplification technique.
Images were initially processed using the MAS 5.0 software (Affymetrix). Hybridization quality was assessed by visual inspection of the image and from a report generated by MAS 5.0. Criteria for an acceptable hybridization were as follows: background < 100, noise (RawQ) < 5, 3'/5' ratio for select housekeeping genes < 4. Hybridizations not meeting these criteria were repeated, beginning at the target preparation step. The data were analyzed and statistically filtered using Rosetta Resolver version 3.0 software (Rosetta Inpharmatics, Kirkland, WA). The threshold for significance was set at p 0.001, and genes which exhibited a 1.5-fold or –1.5-fold change were reported as a fold-change relative to the corresponding control. Similarly regulated genes were visualized using CLUSTER and TreeView (Eisen et al., 1998). Genes were grouped into functional classes with the help of KEGG (http://www.kegg.org) and using Gene Ontology (http://www.geneontology.org) identifiers in the U74Av2 template (version accessed Aug. 1, 2003 from http://www.affymetrix.com). Identification of ESTs was facilitated by euGenes (http://eugenes.org/mouse/).
Real-time RT-PCR analysis.
The levels of expression of the selected genes were quantified using real-time RT-PCR analysis. Briefly, total RNA was extracted as described above and reverse transcribed with MuLV reverse transcriptase and oligo-dT primers. The forward and reverse primers for selected genes (Table 1) were designed using Primer Express software, v2.0 (Applied Biosystems, Foster City, CA). The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis. The relative differences in expression between groups were expressed using cycle time (Ct) values as follows: the Ct values of the genes were first normalized with -actin of the same sample, and then the relative differences between control and treatment groups were calculated and expressed as relative increases, setting the control as 100%. Assuming that the Ct value is reflective of the initial starting copy and that there is 100% efficiency, a difference of one cycle is equivalent to a two-fold difference in starting copy.
Statistics.
Statistical procedures generally followed the scheme of Gad and Weil (1989) using SAS (SAS Institute, Cary, NC) or SigmaStat (Jandel Scientific Software, San Rafael, CA) software. FB1-, CM-, and WY-treated and controls of each strain were compared using ANOVA followed by the Duncan's multiple range test to identify differences among groups (parametric, homogenous data), the Kruskal-Wallis test followed by the distribution free multiple comparisons (nonparametric data) or Fischer's least significant differences test (incidence data). Two-group comparisons of WT and PPAR-null groups receiving the same diet were done by Students t-test. Tests were two tailed, and significance was judged at p < 0.05.
RESULTS
Appearance, Body Weight and Food Consumption
With one exception, a CM-fed PPAR-null mouse that became inactive and not responsive to touch on the last day of exposure, the appearance and activity of the animals were unremarkable. FB1- and CM-fed mice of both strains lost weight (Table 2). Cumulative loss averaged from 2.0 (FB1-fed WT) to 6.5% (CM-fed PPAR-null), but compared to their control groups, statistical significance was found only in FB1- and CM-fed PPAR-null mice. Cumulative weight gain of WY-fed WT mice was significantly increased compared to the WT controls. No significant differences in food consumption were found. Mean daily food consumptions (g/kg body weight) for the WT groups were: control = 237 ± 10.5 (SD), WY = 238 ± 21.5, FB1 = 245 ± 33.9, and CM = 228 ± 15.2. Values for the PPAR-null groups were: control = 232 ± 37.1, WY = 233 ± 26.9, FB1 = 212 ± 31.3, CM = 211 ± 27.2.
Necropsy and Liver Weights
No noteworthy gross lesions were found at necropsy. Final (fasted) body weights of the PPAR-null FB1- and CM-fed groups were significantly reduced (9 to 10%) compared to their controls (Table 3), while no significant differences in final body weight were found among the four WT groups. Both FB1 and the CM significantly increased the absolute (32 to 35%) and relative liver weights (37 to 43%) of WT mice (Table 3). Liver weights of PPAR-null mice fed FB1 or the CM were slightly higher than those of their controls; statistical significance was demonstrated only for the relative liver weight of the CM-fed group (28% greater than the controls). WY significantly increased the absolute and relative liver weights of the WT mice but had no significant effect on liver weights of the PPAR-null animals, as expected.
Microscopic Pathology and Hepatocyte Proliferation
Microscopic appearance of the livers from WT and PPAR control mice was unremarkable. The only observation of note was a mild degree of hepatocellular vacuolation in one WT and all PPAR-null animals. The vacuoles' appearance was consistent with lipid deposition but was not confirmed histochemically. Two to four mice from each of the remaining treatment/strain combinations showed a similar pattern of hepatocellular vacuolation. WY caused marked hepatocytomegaly and cytoplasmic eosinophilia in the WT mice. Densely staining chromatin and obviously increased numbers of mitotic figures were also noted. That WY had no significant effect on the microscopic appearance of livers from PPAR-null mice is consistent with previous observations (Lee et al., 1995; Peters et al., 1997).
FB1 and the CM caused liver lesions in both strains that were consistent with those previously found in fumonisin-exposed mice (Sharma et al., 2002). The lesions included focal apoptosis, mitotic figures, focal oncotic or coagulative necrosis (3–4 mice/group), focal or generalized variation in cell (anisocytosis) and nucleus (anisokaryosis) size (3–5 mice/group), focal acute inflammation (2–3 FB1-treated mice/group), or a slight amount of focal bile duct hyperplasia (2 FB1-treated and 2 CM-treated PPAR-null mice). Overall group mean pathology scores were 1.5 to 1.6 (Table 4), indicating that FB1 and the CM caused a similar degree of liver injury in both strains. The average number of hepatic apoptotic foci per mouse induced by FB1 or CM tended to be slightly higher, and liver mitotic figure counts tended to be slightly lower in the PPAR-null mice (Table 4), but no statistically significant differences for either variable were found between the two strains.
WT mice exhibited significant increases in cell proliferation after exposure to WY, CM, or FB1 as determined using BrdU positive nuclei as the experimental endpoint (Fig. 1). The findings corroborated the histopathology results indicating that cell proliferation was approximately equal in the CM- and FB1-fed WT groups. While PPAR-null mice did not exhibit cell proliferation after WY exposure, cell proliferation rates of FB1- and CM-fed PPAR-null mice did not differ from those of their corresponding WT groups. Thus, histopathology and BrdU results indicate that apoptosis and cell proliferation stimulated by FB1 and CM were PPAR-independent.
Liver Sphingoid Base Concentrations and ACO Protein Expression
Both FB1 and CM increased hepatic Sa concentrations (Fig. 2a) and Sa/So ratios (Fig. 2b) in the WT and PPAR-null strains; So concentrations were unaffected by both. Liver Sa concentrations in the PPAR-null mice fed FB1 were significantly less than those of their WT counterparts. They were also slightly lower in CM-fed PPAR-null than in CM-fed WT mice, but the difference was not statistically significant. However, Sa/So ratios of the groups fed FB1 or CM were not significantly different from one another (8.7 ± 3.1 (SD) for FB1-fed PPAR-null and 12.3 ± 2.6 for FB1-fed WT). WY had no effect on hepatic sphingoid base concentrations in either strain. WY exposure increased the levels of liver ACO and CYP4A in WT but not PPAR-null mice, whereas FB1 and CM did not cause any changes in the expression of these markers of peroxisome proliferation in either strain (data not shown).
Transcriptional Programs Regulated by Fumonisin
We identified 1459 genes that were significantly different between two or more groups (p 0.001; 1.5-fold or –1.5-fold change) as outlined in the Materials and Methods. Most of these genes were altered by WY (1217 genes), consisting of 732 up-regulated and 485 down-regulated genes. FB1 up-regulated 180 genes and down-regulated 193. CM exhibited a similar response, up-regulating 200 genes and down-regulating 203. Many of the genes unique to WY (a total of 1012 genes) have been discussed in previous studies (Anderson et al., 2004a,b; Corton et al., 2004) and will not be discussed here. We focus here on the genes regulated by CM and FB1, including those genes that are also altered by WY exposure.
The genes were classified into major groups based on their expression behavior (Fig. 3). The first two classes were those regulated by CM (56 genes) or FB1 (48 genes) only. The third and fourth classes were genes regulated by CM and WY or FB1 and WY. There were a total of 49 and 27 genes in these classes, respectively. Most of the genes in these classes exhibited similar expression by CM and WY or FB1 and WY. In both classes 33% of the total number of genes exhibited opposite expression behavior. The fifth class of genes consisted of those regulated by both CM and FB1 but not WY. There were 138 genes in this class. The sixth class consisted of genes regulated by all three compounds. There were a total of 160 genes in this category. This class was dominated by genes that exhibited similar regulation by all three treatments (82%) and most of the genes regulated by either CM or FB1 were regulated by both treatments (62%). Furthermore, all of the genes coregulated by CM and FB1 were not only regulated in a similar direction, but the level of up- or down-regulation was similar. These results demonstrate that CM and FB1 exhibit an overlapping set of genes. Furthermore, the majority of genes regulated by CM and FB1 do not overlap with the genes regulated by WY.
The genes regulated by CM or FB1 and WY fell into a number of functional categories including lipid metabolism, cell proliferation, signal transduction, phase I and II xenobiotic metabolism, and glutathione metabolism (Table 5) (Supplementary Table 1 for the complete list). Real-time RT-PCR was used to confirm the expression changes of 27 genes that fell into many of these categories (Table 6). In these experiments we examined the expression changes in WT and PPAR-null mice after compound treatment. In general, the expression changes observed by transcript profiling were corroborated by real-time RT-PCR. However, there were a few exceptions. Fabp1 in WY-treated WT mice exhibited down-regulation by profiling but exhibited up-regulation by real-time RT-PCR, and Cdkn2b in WT mice was down-regulated by all three treatments by profiling, whereas there was up-regulation by CM and FB1 by real-time RT-PCR. CM and FB1 exhibited an identical pattern of changes in WT mice. Almost all of the changes were not only in the same direction but of approximately the same magnitude in WT and PPAR-null mice, demonstrating that PPAR is not required for CM or FB1 to alter gene expression. Exceptions were Cyp4a10, in which CM and FB1 decreased expression in WT but increased expression in PPAR-null mice, and Cdkn2a, which exhibited induction in WT but not PPAR-null mice after FB1 treatment. As expected, WY required PPAR for the majority of changes. PPAR-null mice exhibited some induction of Cyp4a10 and Mte1 after WY exposure. PPAR-independent induction of Cyp4a10 and Cyp4a14 by WY has been observed earlier (Anderson et al., 2004a,b). These findings demonstrate that the gene expression changes after CM or FB1 exposure are PPAR independent and identify novel targets related to FB1 toxicity.
DISCUSSION
There are few studies addressing whether PPAR-dependent signaling pathways are involved in fumonisin toxicity, and the reported results are inconsistent. Gelderblom et al. (1996) concluded that peroxisome proliferation did not play a role in FB1-induced hepatotoxicity in rats, whereas Martinez-Larraaga et al. (1996) observed that FB1 increased CYP4A1 apoprotein levels and -oxidation of palmitoyl coenzyme A and suggested peroxisome proliferation could play a role. FB1 is clearly hepatotoxic (Sharma et al., 2002, 2003) and hepatocarcinogenic (Howard et al., 2001) to female mice; however, its effect on PPAR-dependent pathways in mice is unknown. To address this question, we compared the hepatotoxic effects of FB1 in wild-type (WT) and PPAR-null mice. Fungal culture materials have been used as a fumonisin source in animal feeding studies and have been shown to elicit qualitatively similar morphologic and sphingolipid effects in rodents as FB1 (Voss et al., 2001). However, the fungal culture materials contain other fumonisins and secondary metabolites in addition to FB1 that might exert biological activity (International Programme on Chemical Safety, 2000; Voss et al., 2001). We therefore also compared the hepatotoxic effects of a commonly used F. verticillioides CM in the two mouse stains to determine if the fungus produced any other fumonisins or nonfumonisin compounds having significant PPAR-dependent activity.
The hepatic response of the two strains to WY clearly differed. The absence of liver hypertrophy, hepatocellular cytomegaly, cytoplasmic eosinophilia, mitotic figures, and BrdU positive nuclei in the PPAR-null mice confirmed that PPAR-mediated signaling was disrupted in this strain as seen in earlier studies (summarized in Corton et al., 2000). Contrary to our hypothesis, however, the absence of PPAR-dependent signaling did not significantly abrogate the typical hepatotoxic responses to FB1 or CM. That the expression of ACO, a marker of peroxisome proliferation, and CYP4A was not increased in either the WT or PPAR-null strains is evidence that PPAR-dependent signaling, including induction of peroxisome proliferation, plays no significant role in fumonisin or F. verticillioides hepatotoxicity in mice.
To gain insight into the genes altered by FB1 exposure, we generated transcript profiles from the livers of wild-type mice exposed to CM, FB1, and WY. There was a close correlation in the expression pattern between CM and FB1. Approximately 62% of all genes altered by either CM or FB1 treatments were not only altered in the same direction, but the magnitude of the changes was very similar. In contrast, most of the genes regulated by CM and FB1 do not overlap with those regulated by WY. As both WY and FB1 increase cell proliferation under these conditions, it was not surprising that many of the genes that WY and FB1 have in common are those involved in cell fate, including many involved in the cell cycle. We examined the expression of a number of overlapping genes by RT-PCR in both WT and PPAR-null mice and showed that CM and FB1 alter the expression of genes in a number of different categories independently of PPAR, whereas WY required PPAR for most of the changes. This provides further evidence that FB1 does not act like a typical peroxisome proliferator.
There are only a few reports on fumonisin-induced alteration of gene expression patterns in vivo. Rats fed 250 ppm FB1 for up to 5 weeks exhibited a progressive increase in the expression of several genes involved in cell proliferation and regeneration including hepatocyte growth factor (HGF), transforming growth factor (TGF), and transforming growth factor-1 (TGF-1), as well as the oncongene c-myc (Lemmer et al., 1999). Bondy et al., (2000) found that FB1 increased liver cyclin D1 protein without affecting its mRNA expression in rats treated with FB1 for 6 days. Ramljak et al. (2000) likewise reported a dose-related overexpression of hepatic cyclin D1 in rats fed diets containing up to 250 ppm FB1 for 3 weeks. Overexpression did not result from increased cyclin D1 mRNA transcription but from post-translational stabilization of cyclin D1 through a process dependent on glycogen synthase kinase 3 and Akt. Together, these findings suggested that cell cycle disruption, particularly disruption of G1/S transition, is a critical event in FB1 carcinogenesis. Our results differed from these reports in that cyclin D1 transcription was increased in both mouse strains by fumonisin exposure, whereas the expression of HGF, TGF, TGF-1, or c-myc was not significantly altered. As in rats (Bondy et al., 2000), FB1 and CM increased transcription of the cyclin dependent kinase inhibitor cdkn1A (p21) in mice.
Sharma et al. (2003) found increased expression of mRNA for genes in the livers of mice treated with 2.25 mg/kg body weight FB1 for five consecutive days that are involved in apoptosis (bax, c-myc, caspase 8), cytokine signaling (IL-1 receptor agonist), and tumor necrosis factor (TNF) or Fas signaling (FADD, FAP, FAF, TRAIL, TRADD, RIP, and TNFR1) pathways. In the present study, the expression of two genes involved in apoptosis (clusterin, apoptosis inhibitor 6) was significantly altered by FB1 or CM. The direct and indirect (i.e., mediated by sphingoid base accumulation, decreased ceramide biosynthesis, or complex sphingolipid depletion) effects of fumonisins on gene expression are complex, and determining their mechanistic implications in regard to fumonisin hepatotoxicity or carcinogenicity is beyond the scope of this investigation. However, this list of regulated genes will provide a basis for hypotheses explaining linkages between gene expression changes and phenotypic effects of fumonisins. Future studies should consider dose- and time course-related effects as well as species- and strain-related differences in the expression of gene products. The latter is particularly important when using genetically modified animal models, as demonstrated by the results of Sharma et al. (2003), which showed that FB1 significantly increased the expression of CD95 Ligand (FasL) and other signaling molecules in TNF-null mice compared to their corresponding wild-type controls, thereby offering an plausible explanation for the unexpected increase in severity of hepatotoxicity in FB1-treated TNF-null mice (Sharma et al., 2002).
In summary, results from this in vivo feeding study showed that the hepatotoxic mechanism of FB1 in mice does not involve PPAR-mediated signaling pathways and further indicate that F. verticillioides CM does not contain other metabolites having significant peroxisome proliferator activity. The specific sequence of events linking the inhibition of ceramide synthase by fumonisins to altered gene expression, apoptosis, mitosis, and overt hepatotoxicity in rodents remains to be elucidated.
SUPPLEMENTARY DATA
Supplementary data are available online at www.toxsci.oxfordjournals.org.
NOTES
2 Current address: Bayer Corporation, Research Triangle Park, NC 27709.
3 Current address: Toxicogenomics Program, National Health and Environmental Effects Research Lab, US-EPA, Research Triangle Park, NC 27711.
ACKNOWLEDGMENTS
The authors thank N. Brice, R. de la Campa, P. Malcom, J. Showker, P. Stancel, and E. Wray for their expert assistance. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Disclaimer: Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
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