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ANIMAL PRODUCTION |
* Division of Animal Sciences,
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Veterinary Medical Diagnostic Laboratory, and
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Department of Statistics, University of Missouri, Columbia 65211
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: endophytic toxins • fescue toxicosis • genomic • liver • rat • thermoregulation
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Significant data gaps underlie cellular and metabolic aberrations associated with this problem to limit development of pharmacological strategies. A novel approach to elucidate the altered cellular mechanisms associated with fescue toxicosis is to use microarray analysis (Jones et al., 2004).
The liver is the most important organ for xenobiotic metabolism and detoxification and has been identified as a target organ of fescue toxicosis (Oliver, 1997). Oliver (1997) observed an increase in both glycogenolysis and urine glucose levels in cattle with fescue toxicosis. Piper et al. (1991) noted an increase in liver-specific enzymes in animals grazing endophyte-infected (E+) tall fescue long-term, and a component of the fescue toxicosis response in rats is a decrease in liver weight (Eichen et al., 2001). Chestnut et al. (1992) noted that rats fed an E+ diet exhibited a decrease in liver weight, which was observed in rats fed a control diet at the same caloric level (i.e., pair-fed). Although it is likely that fescue toxicosis has both direct and indirect (i.e., reduced caloric intake) effects on hepatic function, the objective of this preliminary study was to determine the overall impact of fescue toxicosis on hepatic gene expression. It was hypothesized that short-term intake of an E+ diet at thermoneutrality would change hepatic gene expression associated with fescue toxicosis. This research represents one of the first attempts to determine changes in genetic expression associated with fescue toxicosis.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals and Treatment Diet.
All procedures on animals were approved by the University of Missouri Animal Care and Use Committee. Sprague-Dawley male rats (n = 12 per treatment; 42 d old; Charles River Laboratories, Willington, MA), weighing 185 to 215 g, were maintained at 21°C ambient temperature and 50 ± 5% humidity as previously reported (Spiers et al., 2005). Feed and water were provided ad libitum throughout the study. Composition of control and treatment diets were as previously described, with the treatment diet adjusted to provide a specific amount of ergovaline (EV; the major ergot alkaloid associated with fescue toxicosis; Spiers et al., 2005). Control (E–) seed contained 22 ppb of EV, whereas E+ contained a concentration of 4,100 ppb on a DM basis as measured by HPLC (detection limit = 50 ppb and CV = 7%; Rottinghaus et al., 1993).
Treatment Period and Sample Collection.
The experimental schedule consisted initially of an 8-d recovery period after intraperitoneal implantation of telemetric temperature transmitters (Model VM-FH; Mini-Mitter Company, Inc., Bend, OR), followed by a 5-d pretreatment period (d 1 to 5), during which core temperature (Tc), feed intake, and BW were collected to establish a baseline for statistical analysis (Spiers et al., 2005). During the 5-d duration of treatment (d 1 to 5), rats were fed either E– or E+ (91.5 µg of EV·kg of BW–1·d–1) diets, and Tc, feed intake, and BW measurements were collected. At the end of study (d 6), rats were anesthetized by i.m. injection of a xylazine (13 mg/mL)-ketamine (87 mg/mL) cocktail (0.1 mL/100 g) and exsanguinated, and the liver was collected and weighed. A transverse section from the middle lobe of liver (~5 g) was fixed in 10% neutral buffered formalin for histological examination, and another portion (~5 g) was snap-frozen in liquid nitrogen for microarray experiments. Serum from blood obtained at exsanguinations was frozen at –20°C for later use. Serum prolactin concentrations were measured using a rat prolactin enzyme immunoassay kit (SPI-BIO, Massy Cedex, France) as previously reported (Duhau et al., 1991).
Histological Examination of Liver. Fixed liver samples were embedded in paraffin, and 4-µm sections were stained with hematoxylin and eosin (H&E) to study histological changes and with periodic acid-Schiff’s reagent to compare the levels of stored glycogen in E+ and E– rats. Images were captured at 400x magnification (Insight Color Camera, Sterling Heights, MI). A scoring system (1 to 4) was devised to quantify glycogen storage; 1 denoted little or no vacuolation and 4 indicated marked vacuolation. A vacuolation score was assigned to the liver sections by a board-certified veterinary pathologist without prior knowledge of the treatment group assignments.
Statistical Analysis of Physiological Responses. Mean values for Tc, feed intake, and BW were computed as a function of time and treatment group. These parameters were analyzed by repeated measures ANOVA (Mixed procedure) of SAS (SAS Inst., Inc., Cary, NC) using treatment, time, and animal as class variables to determine overall treatment and time-related differences, and treatment x time interactions.
Microarray and Real-Time PCR Analyses of Genomic Responses
Selection of Experimental Animals. In the current study, 8 out-bred rats (4 per treatment group) were used to study the genomic effects of fescue toxicosis. Because of the costs associated with microarray analyses, 4 E+ rats showing the most pronounced evidence of fescue toxicosis were selected for microarray analysis. Selection was based on reductions in feed intake, FCE, liver weight, and Tc. Quantitative real-time PCR (RT-PCR) was used for all 24 rats to validate the microarray results at the population level.
Microarray Slide Printing. Microarray slides were prepared by printing 1,353 oligonucleotides (50 bases long) specific to rat liver (MWG Biotech AG, Ebersberg, Germany) on pan epoxy glass slides (MWG Biotech AG) using a 16-pin robotic microarray printer. Each block in the microarray slide also contained: 10 Arabidopsis thaliana plant genes (Spot report oligo, Stratagene, Cedar Creek, TX) as external controls, 3x SSC as negative controls, and rat Cot-1 DNA (Stratagene) and poly A (Stratagene) to detect nonspecific hybridization.
RNA Extraction.
Total RNA was extracted from the liver samples using an RNeasy Midi Kit (Qiagen Inc., Valencia, CA), purified using DNase-1 (Ambion Inc., Austin, TX) and phenol:chloroform:isoamyl alcohol (25:24:1), and concentrated using Microcon YM30 filters (Millipore Corp., Bedford, MA), and following the manufacturer’s instructions. The RNA concentrations were measured using an ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE; Flanagan, 2005).
cDNA Preparation. Concentrated E+ or E– total RNA (15 µg) was reverse-transcribed to cDNA using oligo dT (IDT DNA, Coralville, IA), random hexamers (IDT DNA), 50x amino-allyl dUTP (Sigma Chemicals, St. Louis, MO), and reverse transcription (StrataScript RT, Stratagene, La Jolla, CA). The resulting cDNA was purified using Microcon-30 filters (Millipore) and conjugated with either Cy3 or Cy5 dyes separately by incubating in the dark for 1 h. Conjugated oligonucleotides were separated from free, unconjugated dye using a Qia-quick PCR purification kit (Qiagen) and following the manufacturer’s instructions. The 2 cDNA samples, labeled with Cy3 and Cy5 dyes, were mixed together and dried.
Microarray Hybridization. The dried cDNA pellet was resuspended with the hybridization mixture, which consisted of 1x 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (pH 7.0), poly A (10 µg/µL; Stratagene), rat Cot-I DNA (3.2 µg/µL), 20x standard saline citrate (SSC; Stratagene), and 10% sodium dodecyl sulfate. The slides were incubated in a water bath (65°C) for 12 to 16 h. After incubation, the slides were rinsed in wash solution I (20x SSC, and 10% sodium dodecyl sulfate) and then in wash solution II (20x SSC).
Microarray Slide Scanning and Analysis.
Microarray slides were scanned using a GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA). The signal intensity of each spot of the scanned image was analyzed using Genepix Pro 3.0 software (Axon Instruments). The spots were filtered based on their spot intensities, and flagged as unusable if the number of pixels that exceeded a background-related threshold (background intensity ± 1 SD) was lower than 55%. The spots were flagged as not found when the spot had less than 6 pixels above the background-related threshold, or if the spots overlapped the adjacent spots. The microarray data were stored in the BioArray Software Environment database (Saal et al., 2002).
Self-Self Hybridization. Six self-self hybridizations were performed to measure the variation in gene expression due to technical errors. In these, the same rat was used for different RNA isolations, labeling, and hybridizations. Slides were hybridized, scanned, and analyzed as just described. Means and standard deviations for Cy5 and Cy3 ratios were calculated for the 6 hybridizations, and a significant threshold was set as mean ± 2 SD (i.e., ± 1.34). Genes with expression above or below this threshold were considered as up- or downregulated, respectively, and were used for further analysis.
Microarray Analysis Using an ANOVA Model.
A loop design was followed for the microarray experiments. Intensity readings were transformed to log base 2 scales. The microarray data were analyzed using a 2-step mixed linear model following the procedures of Kerr and Churchill (2002) and Wolfinger et al. (2001). The first step was normalization across genes, which identified and removed the variation caused by the array, pin, and dye effects, and all of their first order interactions. The residuals from this model were used as the input for the second stage to model and remove the animal and dye effects and their interactions. The residual values from these analyses represented the normalized data set. Use of the ANOVA model allowed the experimental effects to be estimated through the partitioning of the variations in the experiment and helped in intertreatment comparisons without an internal reference.
First model (normalization):
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in which
adgp
The residual values from the normalization model were obtained by subtracting the fitted value for the effects from the Yagp value. Variation due to pin and array effects significantly contributed to gene expression (25%).
The second model of the microarray analysis consisted of a gene-specific model for the output from the normalization model.
Second model (gene-specific model):
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in which
dgn
Microarray analyses were computed using the Proc Mixed procedure of SAS. A t-test was used to find differentially expressed genes, between treatment groups. Residual values obtained after the normalization model were also analyzed using a nonparametric Kruskal-Wallis method. Similar P-values were obtained for the differentially expressed genes using either method.
The gene by animal values obtained from the ANOVA model analysis were further analyzed using the significance analysis of microarray (SAM; Tusher et al., 2001) program. Microarray data were permutated, using the following parameters: 2-class, unpaired, not log transformed, 1,000 permutations. A hierarchical tree was built with results from the SAM analysis using the methods of Eisen et al. (1998). The hierarchical tree was confirmed using bootstrap (Kerr and Churchill, 2002). The DAVID (Dennis et al., 2003) and Locuslink (http://www.ncbi.nlm.nih.gov/LocusLink) were used to find the gene annotation and the functions of differentially expressed genes.
Microarray raw data files were deposited with the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo/). The serial entry number for the microarray data is GSE2452, and the platform number is GPL 1786. Each hybridization in the microarray experiment was given the following serial numbers: GSM46406, GSM46407, GSM46408, GSM46409, GSM46410, GSM46411, GSM46412, GSM46413, GSM46414, GSM46415, GSM46416, GSM46417, GSM46418, GSM46419, GSM46420, GSM46421, GSM46422, GSM46423, GSM46424, GSM46425, GSM46426, GSM46427, GSM46428, and GSM46429.
Quantitative Real-Time PCR.
Real-time PCR was performed in a subset of 8 representative genes for which the fescue toxicosis-induced changes in expression were considered statistically significant by microarray analysis, and the pathways represented were of interest from the perspective of fescue toxicosis-induced hepatic effects. Table 1 shows the gene-specific primers that were designed with the Primer Express program (Applied Biosystems, Warrington, UK) using the default settings (primer Tm = 58 to 60°C; GC content = 30 to 80%; length = 9 to 40 nucleotides). Glyceraldehyde-3-phosphate dehydrogenase was used as the housekeeping gene. The annealing temperature for the primers was standardized using gradient PCR and 3% agarose gels. The reaction mixture consisted of 2x SYBR Green PCR Master mix (Applied Biosystems), 50 nM of forward and reverse primers, and 0.1 ng of cDNA/µL. Thermal cycling was carried out with an ABI Prism 7500 sequence detection system (Applied Biosystems) under factory default conditions (50°C, 2 min; 95°C, 10 min; and 40 cycles at 95°C for 15 s, and 60°C for 1 min). Each gene was measured in triplicate, and the formation of a single PCR product was confirmed using dissociation curves. The normalized Ct values for each gene from all of the 12 rats in each treatment group were averaged, and the relative percentage change in the expression of the gene in E+ rats was calculated. Student’s t-test was used to compare the Ct values between E+ and E– rats.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Core Body Temperature.
A previously reported (Spiers et al., 2005) circadian rhythm for the rat Tc was also observed in this study. Consumption of E+ diet resulted in a time-related decrease (P = 0.04) in Tc (Figure 1) in the E+ fed treatment group, with the temperatures decreasing (P < 0.05) from d 4. In contrast, no significant decrease in Tc was observed in E– rats during the treatment period.
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No significant changes were observed in mean FCE in E– rats during the treatment period. However, average FCE dropped in the E+ rats (P < 0.01) immediately after the first day of treatment (–14.97 g of feed/kg of BW). By the end of d 5, there was an increase (P = 0.02) in average FCE in E+ rats compared with d 1 during the treatment period. Despite this improvement, average FCE was still much lower in the E+ treatment group (–11.09 g of feed/kg of BW) compared with the E– treatment group (32.24 g of feed/kg of BW) at the end of treatment (P < 0.01).
Body and Organ Weights. Mean BW of E+ rats was decreased (P < 0.05) on d 4 and 5 compared with E– rats; the average BW of E– and E+ rats was 333.26 ± 8.11 and 295.57 ± 8.11 g, respectively, at the conclusion of the study. Mean liver weights were compared between E+ and E– rats as a preliminary measure of the effect of fescue toxicosis. Average wet weights of liver in g/kg of BW decreased in E+ (38.43 ± 0.15) compared with E– (44.55 ± 0.15) rats (P < 0.01).
Additional Effects.
Liver sections stained with H& E showed differences with respect to the amount of cytoplasmic vacuolation in E+ rats compared with E– rats (Figure 2). In the H&E-stained hepatic sections, the number of vacuoles differed (P < 0.01) in E– (2.58) compared with E+ (1.67) rats suggesting decreased feed intake or anorexia in E+ rats. The vacuoles represent hepatic stores of glycogen and fat. Qualitative staining with periodic acid-Schiff’s reagent suggested decreased stores of glycogen in the livers of E+ relative to E– rats because of the decrease in dark pink cellular inclusions (Figure 2). As expected, the mean serum prolactin concentration was lower (P = 0.02) in E+ rats (6.05 ± 0.51 ng/mL) as compared with the E– rats (12.95 ± 1.28 ng/mL), verifying the existence of fescue toxicosis in the E+ treatment group.
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Hepatic Microarray Results.
Average mean ± 2 SD intensity values (±1.34) of the 6 self-self hybridizations were used as the threshold to exclude the genes that exhibited small changes in gene expression but were identified as significant (P < 0.05) because of unusually small variance. Microarray analysis using the SAM program showed 122 genes being differentially expressed with a false discovery rate (FDR) of less than 32%. The functions of 19 differentially expressed genes are unknown (data not shown). These genes are shown in Tables 2 to 8. A large proportion of these differentially expressed genes (93) were downregulated in the E+ treatment group, as compared with E– treatment group, with the expression of the remaining 29 genes being upregulated in the E+ rats relative to the E– group. Hierarchical clustering showed the differentially expressed genes in the rat hepatic tissue (Figure 3), and bootstrap testing supported the hierarchical tree results with 100% confidence. Important pathways represented by the differentially expressed genes included energy metabolism, growth and development, detoxification and antioxidant mechanisms, as well as stress response, immune function, cell integrity and transportation, and other cellular processes.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fescue toxicosis in cattle is associated with a number of physiological responses, including altered thermoregulation (Stuedemann et al., 1985; Spiers et al., 1995), decreased feed intake and average daily gain (Nihsen et al., 2004), inefficient feed conversion (Bush et al., 2001), and hypoprolactinemia (Schillo et al., 1988; Cross et al., 1995). All except the last 2 responses have been previously reported in the rat model for fescue toxicosis (Spiers et al., 2005). However, all of these responses were observed in the current study after only 5 d of exposure to an E+ diet at thermoneutrality (Figures 1
, 4, and 5).
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-adrenergic, dopaminergic, and nearly all subtypes of serotinergic receptors (Oliver, 2005). Interactions of E+ toxins with these receptors increase vasoconstriction (Oliver, 2005), decrease feed intake, gastrointestinal secretions, segmental activity, nutrient absorption (Oliver, 1997), and suppress prolactin secretion from the anterior pituitary (Elsasser and Bolt, 1987). Genomic Responses Associated with Fescue Toxicosis
Short-term intake of an E+ diet at thermoneutrality was hypothesized to result in changes in hepatic gene expression that is associated with the pathogenesis of fescue toxicosis. Microarray and real-time PCR results demonstrated that the expression of genes belonging to several important physiological pathways, including energy metabolism, growth and development, detoxification and antioxidant activity, as well as stress response and immune function, were altered by E+ treatment (Figure 6). Genomic responses to the direct hepatic effects of endophytic toxins or, secondarily, to indirect effects of fescue toxicosis on the liver (e.g., decreased caloric intake) are of interest to scientists looking for novel prophylactic and therapeutic approaches to endophyte-infected fescue problems in livestock. A careful analysis of these genomic effects and their relationship to the observed physiological responses should provide additional insight into the pathophysiology of fescue toxicosis.
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), all of these were also observed in the current study (Figures 4 and 5). Hepatic effects of these physiological responses were demonstrated by both the decreased mean relative liver weight of E+ compared with E– rats, which was also observed by Chestnut et al. (1992) in rats pair-fed an E– diet, and by histological changes in the E+ livers, which most likely reflected depletion of hepatocellular stores of glycogen and lipids (Figure 2). Consistent with decreased caloric intake and FCE, genes involved in lipid and carbohydrate metabolism (e.g., liver-specific Bhlh-Zip transcription factor-7, ATP citrate lyase, and glucuronidase beta) were downregulated by E+ treatment (Table 2). Fescue toxicosis-induced upregulation of genes involved in gluconeogenic pathways, such as phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase (Table 2), indicated an adaptive response to lower caloric intake during exposure to E+ diets. In addition, several genes associated with protein catabolism in other pathways, including arginase, 4-hydroxy phenyl pyruvic acid dioxygenase, and epoxide hydrolase 1, were also upregulated in E+ rats (Tables 3 and 4), supporting a relationship between protein catabolism for gluconeogenesis in E+ rats. Other studies have shown that during starvation, genes involved in gluconeogenesis (e.g., phosphoenolpyruvate carboxykinase; Perez et al., 1998), and protein catabolism (e.g., 4-hydroxy phenyl pyruvic acid dioxygenase; Endo, 1999), are upregulated.
Several important genes involved in mitochondrial activity (i.e., ATP production) were also downregulated in E+ compared with E– rats (Table 2). The gene for mitochondrial transcription factor A encodes for a key activator of mitochondrial transcription, as well as a participant in mitochondrial genome replication. Mitochondrial transcription factor A is required for accurate and efficient promoter recognition by mitochondrial RNA polymerase (Murthy et al., 1981). Mitochondrial ATP synthase is mainly involved in production of ATP from ADP in the presence of a proton gradient across the mitochondrial membrane (Yoshida et al., 2001). Downregulation of these important genes, as well as ATPase 3 (proteosome 26S subunit) and thioredoxin reductase 2 (Table 4), in E+ rat livers could impair ATP production and would help explain the reduced FCE and hypothermia at thermoneutrality observed in endophyte-exposed animals.
Growth and Development.
Average daily BW was consistently lower throughout the treatment period in E+ rats (Figure 4) despite the apparent recovery in average daily feed intake of these animals (Figure 5). This lack of recovery in feed intake of E+ rats reflected the effects of endophytic toxins, decreased caloric intake, and reduced FCE. The somatomedins or IGF mediate many of the growth-promoting effects of growth hormones. In association with reductions in FCE and ADG, genes for IGF-I, IGFBP-2, and other genes involved in body growth and development, such as erv1-like growth factor (hepatopoietin), growth response protein, and eukaryotic translation initiation factor 4E binding protein-1, were downregulated in E+ as compared with E– rats (Table 3).
Apoptosis or programmed cell death occurs during development and aging and can be beneficial. However, excessive apoptosis can result in tissue damage. In the current study, genes that protect the cell from excessive apoptosis, such as IGF-I (Mason et al., 2000), IGFBP-2, inhibitor of apoptosis protein-1 (Verhagen et al., 2001), heme oxygenase 1 (Brouard, 2000), and CCAAT/enhancer binding protein, were downregulated in E+ compared with E– rats (Table 3). Downregulation of these genes has the potential to increase the rate of apoptosis in E+ exposed hepatic tissue.
Detoxification and Antioxidant Activity.
Hepatic biotransformation of xenobiotics (phase I metabolism) and the subsequent conjugation of the resulting metabolites into compounds that can be excreted from the body (phase II metabolism) are the primary hepatic processes involved in detoxification. Phase I metabolism frequently involves the cytochrome P450 (CYP) system, which is comprised of many mixed function oxidative enzymes. The CYP enzymes are membrane-bound proteins that catalyze oxidation of many endo- and xenobiotic compounds. CYP 2E1 and CYP 2C13 play important roles in xenobiotic metabolism (Oguri and Yamada, 1994), and livers from E+ rats showed upregulation of the genes for these 2 enzymes when compared with the livers from E– rats (Table 4). The gene for epoxide hydrolase-1, another important enzyme involved in detoxification (Reid et al., 2002) was also upregulated in the livers of E+ rats (Table 4).
Increased expression of these detoxifying genes might assist in the metabolism of endophytic ergot alkaloids, thereby reducing some of the clinical signs of fescue toxicosis, and this proposed detoxification could be one explanation for the apparent recovery in feed intake observed in E+ rats in the current study (Figure 5). Other studies have noted CYP induction after exposure to ergot alkaloids and have proposed a role for this enzyme system in the detoxification of ergot alkaloids. Zanzalari et al. (1989) observed a 70% increase in hepatic mixed-function oxidase activity in E+ ewes after an 11-d endophyte-infected fescue feeding trial. Moubarak and Rosenkrans (2000) observed hydroxylation of ergotamine by a CYP isoform CYP3A4 in beef hepatic microsomes, and Ball et al. (1992) observed the role of CYP3A4 in the initial metabolism of ergot alkaloids via deethylation in human liver microsomes.
Interestingly, upregulation of genes belonging to the CYP system can also be associated with decreased feed intake. Bauer et al. (2004) noted that various CYP isoforms were differentially expressed in murine livers during starvation. Therefore, it is likely that the CYP gene upregulation observed in E+ rats in the current study could be the result of both endophytic toxin exposure and a reduction in caloric intake.
Production of unstable intermediate metabolites during phase I oxidation often leads to the production of free radicals (Scanlan, 2001). These reactive intermediates from phase I metabolism undergo conjugation (e.g., sulfation, glucuronidation, glutathionation, methylation, and acetylation) mediated by phase II enzymes to produce extremely hydrophilic compounds that can be excreted (Scanlan, 2001). Glutathione synthase is involved in a variety of biological functions, including protection of cells from oxidative damage by free radicals, detoxification of xenobiotics, and membrane transport (Grant et al., 1997). A variety of other enzymes are also involved in preventing oxidative damage in the liver. Heme oxygenase is a microsomal enzyme that catalyzes the oxidation of heme to the antioxidant molecules, biliverdin (Maines, 2000). Propyl 4-hydroxylase alpha neutralizes superoxides (Aravind and Koonin, 2001), and thioredoxin reductase-2 reduces disulfide in oxidized thioredoxin to a redox-active disulfide. Decrease in the disulfide reducing activity of thioredoxin reductase-2 leads to a large increase in the NADPH oxide activity, thereby producing more free radicals (Arner et al., 1995).
Microarray analysis in the current study showed that the genes involved in glutathione biosynthesis and glutathione synthetase were downregulated in E+ rat liver (Table 4). Additional genes having antioxidant activity, such as heme oxygenase 1, thioredoxin reductase-2, propyl 4-hydroxylase alpha subunit, and peroxiredoxin-3 were also downregulated in E+ compared with E– rats (Table 4).
Downregulation of the genes for antioxidants in the current study could lead to the accumulation of free radicals within the cell and is consistent with the results of earlier studies. Lakritz et al. (2002) observed lowered blood levels of glutathione and feed intake during fescue toxicosis in cattle under heat stress conditions. Oliver (1997) observed a decrease in the mean plasma antioxidant capacity in E+ steers 7 times below that of E– animals. Oliver (1997) hypothesized that the reactive oxygen species might contribute to the inflammatory response in E+ fed animals.
It is unclear whether the downregulation of genes associated with antioxidant activity in the liver in E+ rats is a direct effect of endophytic toxins or is secondary to a reduction in antioxidant precursors associated with decreased feed intake and FCE. Regardless, decreased antioxidant activity in the liver of endophyte-exposed animals is a possible mechanism for some of the clinical signs associated with fescue toxicosis. This potential for fescue toxicosis-associated oxidative stress is enhanced if phase I enzymatic activity is high while the activities of phase II enzymes and other hepatic antioxidants are low in endophyte-exposed animals (Scanlan, 2001). Therefore, the upregulation of genes for Phase I enzymes and the downregulation of genes involved in antioxidant activity in fescue toxicosis could lead to the accumulation of free radicals within the cells due to the impairment of cellular anti-oxidative mechanisms.
Stress Response.
Acute phase proteins are serum proteins that increase in animals under stress conditions (Lopez-Hellin et al., 2005). Genes involved in acute phase response such as bile acid coenzyme A, bile acid coenzyme ligase A, and coagulation factor-2 were upregulated in the livers of E+ rats, as compared with the livers of E– rats (Table 5). Heat shock proteins, which are usually upregulated in response to stress to protect cellular proteins (Gonzalez and Manso, 2004), were actually downregulated in the current study in E+ compared with E– rats. These downregulated heat shock protein genes in E+ rats included small stress protein, crystalline alpha B, and serine proteinase (Table 5). Fescue toxicosis has been reported to increase rat susceptibility to various stressors. In 2 separate studies, Filipov et al. (1999a, b) observed greater susceptibility of E+ mice to subcutaneous injections of cytokines and greater susceptibility of cattle grazing on E+ pastures to the effects of lipopolysaccharides than their respective E– cohorts.
Immune Function.
Several genes associated with immune function, such as tubulin beta-5, scavenger receptor class B member 1, interferon beta 1, interleukin receptor accessory protein, and tubulin tyrosine ligase were downregulated in the livers of E+ rats compared with the livers of E– rats (Table 6). Altered immune responses and inflammatory changes are commonly observed in fescue toxicosis (Filipov et al., 1999a). Rodents fed E+ toxins exhibited lowered red and white blood cell counts, reduced response to mitogens, and increased T suppressor cell numbers in the spleen compared with E– rats (Dew et al., 1999).
Cellular Integrity, Transportation, and Other Processes.
Twenty-five genes having to do with cell integrity and transportation, as well as another 25 genes involved in other cellular processes, were differentially expressed in the livers of rats fed E+ diets (Tables 7 and 8). Of these 50 differentially expressed genes, only 7 genes involved in structural and membrane transport processes were upregulated. Given the relative lack of documentation of the effects of fescue toxicosis on these pathways, it is difficult to comment at length on the possible role of these genes in the pathogenesis of fescue toxicosis. However, future studies with pair-fed animals will help to determine whether the differential hepatic expression of these genes is related to the direct effects of endophytic toxins or to secondary effects on the liver associated with the reduction in caloric intake.
The current study provides information regarding specific hepatic genes, which are differentially expressed in rats after intake of E+ diets at thermoneutrality. It has also helped to elucidate the cellular mechanisms involved in the pathogenesis of fescue toxicosis. Of particular interest are the pathophysiological roles of reduced caloric intake and FCE, as well as oxidative stress, in the development of signs of fescue toxicosis. Results of this study emphasize the importance of the inclusion of pair-fed treatment groups in future studies to differentiate the direct hepatic effects of endophytic toxins from the secondary effects on the liver that are associated with decreased feed intake. Future studies should be designed to identify differentially expressed genes during various time periods after exposure to endophyte-infected fescue and heat stress, to determine specific markers to be targeted for prophylactic interventions, therapeutic interventions, or both.
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Footnotes |
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2 Corresponding author: spiersd{at}missouri.edu
Received for publication July 14, 2005. Accepted for publication December 5, 2005.
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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