title: Differential gene expression of ewes varying in tolerance to dietary nitrate.
abstract: Ruminants consuming diets with increased concentrations of nitrate (NO(3)(-)) can accumulate nitrite (NO(2)(-)) in the blood, resulting in toxicity. In a previous experiment, ewes identified as highly tolerant to subacute dietary NO(3)(-) were able to consume greater amounts of NO(3)(-) than lowly tolerant ewes without exhibiting signs of toxicity. We hypothesized that highly tolerant and lowly tolerant ewes differ in their ability to metabolize NO(3)(-) and thereby differ in the expression of hepatic genes involved in NO(3)(-) metabolism. Therefore, our objective was to identify hepatic genes differentially expressed between ewes classified as lowly tolerant and highly tolerant after administration of a subacute quantity of dietary NO(3)(-). Analysis of the Bovine Oligonucleotide Microarray data identified 100 oligonucleotides as differentially expressed (P < 0.05) between lowly tolerant and highly tolerant ewes. Functional analysis of the genes associated with these oligonucleotides revealed 2 response clusters of interest: metabolic and stress. Genes of interest within these 2 clusters (n = 17) and nonclustered genes with the greatest fold changes (FC; n = 5) were selected for validation by real-time reverse-transcription PCR. Relative expression, genomic regulation, and FC agreed between microarray and real-time reverse-transcription-PCR analyses, and FC differences (P < 0.05) between lowly tolerant and highly tolerant ewes were confirmed for 12 genes. Metabolic genes that were downregulated (P ≤ 0.032) in lowly tolerant ewes vs. highly tolerant ewes included aldehyde oxidase 1, argininosuccinate lyase, putative steroid dehydrogenase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase1, and sterol carrier protein 2. In contrast, the metabolic gene homeobox was upregulated (P = 0.037) in lowly tolerant ewes. The glutathione peroxidase 3 and inter-α (globulin) inhibitor H4 genes in the stress response cluster were upregulated (P ≤ 0.045) in lowly tolerant ewes. Genes with the greatest FC, but did not cluster within the functional analysis included haptoglobin, which was upregulated (P = 0.024) in lowly tolerant ewes, and fatty acid desaturase 2 and thyroid hormone responsive, both of which were downregulated (P ≤ 0.019) in lowly tolerant ewes. Results from this study indicate that hepatic gene expression differs in ewes identified as lowly tolerant and highly tolerant to increased dietary NO(3)(-).
authors.affiliation: Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Cockrum
authors.forename: K J
authors.initials: KJ
authors.lastname: Austin
authors.forename: J W
authors.initials: JW
authors.lastname: Kim
authors.forename: J R
authors.initials: JR
authors.lastname: Garbe
authors.forename: S C
authors.initials: SC
authors.lastname: Fahrenkrug
authors.forename: J F
authors.initials: JF
authors.lastname: Taylor
authors.forename: K M
authors.initials: KM
authors.lastname: Cammack
mesh_terms: D000818:Animals; D002417:Cattle / Q000235:genetics; D004032:Diet / Q000662:veterinary; D004361:Drug Tolerance / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D005819:Genetic Markers / Q000235:genetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology* / Q000633:toxicity; D020411:Oligonucleotide Array Sequence Analysis / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep / Q000235:genetics* / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D009566:Nitrates