3 research outputs found
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Validation of a novel extraction method for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation by ruminal microbiota
A simple, fast liquid-liquid extraction method was developed for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation using small sample volumes. The method was tested
in vitro with anaerobic incubations of RDX with whole rumen fluid (WRF) and a commercial S.
acetigenes strain in methanogenic media for RDX. Additionally, validation experiments were
conducted in deionized water in order to show applicability towards various aqueous matrices.
Conditions for extraction were as follows: 300 ÎĽL of sample were mixed with an equal volume
of a 0.34 M ammonium hydroxide solution to reach a basic pH, extracted with a hexane/ethyl
acetate 1:1 (v/v) solution (1 mL) and shaken vigorously for 10 secs. The resulting organic phase
was transferred, then dried under a constant flow of Nâ‚‚ and reconstituted with acetonitrile
(300ÎĽL) for HPLC- UV and LC-MS/MS analysis. Percent recovery values were obtained (83-101%) in all matrices for RDX. In WRF (n =3 animals), RDX degradation was observed with
almost 100% elimination of RDX after 4 h. The five nitroso and ring cleavage metabolites (were
observed by mass spectrometry. Liquid cultures of S. acetigenes did not show significant RDX
biodegradation activity. Deionized water extractions indicated acceptable recoveries with low
variability, suggesting suitability of the method for aqueous matrices. Overall, the new method
demonstrated acceptable efficiency and reproducibility across three matrices, providing an
advantageous alternative for studies where complex matrices and small volume samples are in
use.Keywords: HPLC-UV, Biodegradation, RDX, Organic extraction, Ovine ruminal microbiot
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Bovine hepatic differential gene expression in response to perennial ryegrass staggers
“Perennial ryegrass staggers” is a neurological condition characterized by muscular tremors and ataxia in livestock that ingest the indole diterpene lolitrem B from endophyte-infected perennial ryegrass. While the neurotoxic mechanism of action of ryegrass staggers has been defined, little is known about metabolic and other molecular processes that lolitrem B may affect in livestock. The objective of this study was to characterize differential gene expression in the liver of animals fed lolitrem B-containing feed over an extended period of time. Eighteen steers were fed one of three rations (n =6/group) containing lolitrem B (247, 1,554 or 2,256 parts per billion (ppb, DM basis)) over 64 days in a double-blind study. Microarray and confirmatory qPCR were performed to evaluate the hepatic gene expression profile from biopsies taken on days 0 and 64. Widespread perturbation of gene expression was observed in all groups receiving lolitrem B, with a total of 152 differential genes identified (FDR ≤ 0.05). This suggests that chronic exposure to lolitrem B, even at levels below the current threshold of toxicity (2,000 ppb LB), can perturb many genes, biological processes and pathways. Gene ontology (GO) and Kyoto Encyclopedia of Genes and genomes (KEGG) pathway analyses indicated that many of these genes were categorized under lipid/steroid biosynthesis/metabolism and oxidation-reduction. Specifically, genes involved in the biosynthesis pathway of ceramide, a sphingolipid molecule (ACSS2, LASS6 and SCD) and changes in neurosignaling through alteration of nitric oxide synthase activity (ARG1 and GPX4) were up-regulated. Future work should focus on the overall balance between ceramide and its metabolites and antioxidants/oxidants in a variety of body matrices in animals with perennial ryegrass staggers, to determine how these compounds contribute to the overall etiology of this disease.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Wageningen Academic Publishers. The original publication is available at: http://www.wageningenacademic.com/doi/10.3920/WMJ2014.1749Keywords: microarray, lolitrem B, cattle, ceramid