ABSTRACT
Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.
Subject(s)
Cytochrome P-450 Enzyme Inducers/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Gene Expression Profiling , Immunoassay , Liver/drug effects , Nanotechnology , Transcriptome , Xenobiotics/metabolism , Animals , Biotransformation , Cytochrome P-450 Enzyme Inducers/toxicity , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/immunology , Enzyme Induction , Female , High-Throughput Nucleotide Sequencing , Humans , Liver/enzymology , Male , Proof of Concept Study , Rats, Wistar , Reproducibility of Results , Substrate Specificity , Toxicokinetics , Workflow , Xenobiotics/toxicityABSTRACT
A male-specifically expressed sequence tag was used as a probe to screen adult male Oesophagostomum dentatum (Nematoda; Strongylida) gene libraries. The cDNA clones isolated coded for a serine/threonine protein phosphatase with approximately 85% identity to two Caenorhabditis elegans proteins implicated in reproduction. However, the genomic structures for the two species were distinct, in that the O. dentatum gene contained seven introns, whereas the C. elegans homologues contained three (two of which were conserved between the two nematodes). The promoters of all three nematode genes contained two putative GATA motifs separated by six to seven nucleotides and located within 100 nucleotides of the predicted transcriptional start site. RNA interference (RNAi) experiments in C. elegans, targetting the two homologues, revealed a consistent reduction in the number of progeny produced by treated worms, indicating a functional role in reproduction. Expression of green fluorescent protein, directed by the putative promoters for the C. elegans phosphatase genes, was analysed in transgenic C. elegans. The present results suggest that there is a significant degree of conservation between O. dentatum and C. elegans in the features and function of the serine/threonine protein phosphatase characterised, which should have implications for detailed investigations into molecular reproductive processes of some parasitic nematodes.
