Drug metabolizing enzymes and transporters in bos taurus and cryopreserved pig hepatocytes: constitutive expression and transcriptional modulation

The exposure to xenobiotics may alter the transcription of a broad array of genes expressed in multiple tissues and vital organs such as the liver, kidney, intestine, lungs, brain, placenta, and pancreas, in order to modulate their own metabolism and excretion (Tolson & Wang, 2010). The mammalian nuclear receptor superfamily of transcription factors is involved in the first step of detoxification mechanism, regulating the gene expression of phase I and II metabolizing enzymes and drug transporters (di Masi et al., 2009). Over the past ten years, remarkable advances have been made about the nuclear receptor-dependent activation of drug metabolizing enzymes and their consequences. Despite this, such a knowledge is still restricted to human and rodent species and much less is known in veterinary species. Aim of the present PhD thesis, which is presented in form of a collection paper, was first to propose a new nomenclature for bovine cytochrome P450 sequences involved in xenobiotic metabolism through a phylogenetic analysis and, thereafter, to improve knowledge on distribution and regulation of genes involved in xenobiotic metabolism of cattle and pig, two of the most important food-producing species. For this reason the transcriptional effects of phenobarbital were investigated upon bovine hepatic and extra-hepatic tissues nuclear receptors, drug metabolizing enzymes and major uptake and efflux transporters. Furthermore, transcriptional and post-translational effects of culturing time and prototypical cytochrome 3A inducers were investigated on principal transcription factor and metabolizing enzyme coding genes in long-term stored cryopreserved pig hepatocytes. The phylogenetic analysis pointed out several inconsistencies between currently used cattle cythochromes P450 names and true evolutionary relationship among cytochrome P450 isoforms. Name changes mostly mirrored the orthologous counterparts established for other species, and new names were created when no clear orthologous sequences were identified. With regard to drug metabolizing enzymes and drug transporter expression in cattle tissues, the obtained information confirm for the most part data obtained in humans and rodent model species. Therefore, even if present results are not suggestive of a clear bovine-specific pattern of gene expression, the existence of a tissue-specific mechanism of cythochromes P450 induction cannot be excluded. Phenobarbital, orally administered at inducing dosages, up-regulated cattle hepatic cytochrome 2B, 2C and 3A, as well as the major conjugative enzymes UDP glucuronosyltransferase 1A1-like and glutathione S-transferase α 1-like. In extrahepatic tissues, results showed a different responsiveness to phenobarbital when compared to liver. A general trend to inhibition was observed in small intestine, and the sulfotransferase 1A1-like gene expression was significantly inhibited. About drug transporters, liver, intestine and testis were proved to be tissues mostly involved in transport activity and, ATP-binding cassette C2 mRNA was shown to be significantly up-regulated by phenobarbital in the liver. By contrast, renal ATP-binding cassette G2 mRNA was inhibited. Likewise to former studies made in primary or cryopreserved hepatocytes obtained from different species (including the pig, too), a time-dependent down-regulation of cytochrome mRNAs and proteins was observed, whereas nuclear receptor gene expression data showed some differences in terms of response (crash and rise effect, up-regulation, no effect). When exposed to prototypical cytochrome 3A inducers, both nuclear receptor (essentially following the incubation with dexamethasone) and cytochromes gene expression profiles were up-regulated; furthermore, phase I enzymes modulation was confirmed at the post-translational level, although to a lower extent. In conclusion, the gene expression profiling represents nowadays a helpful and sensitive method, compared to protein and catalytic activity assays, to detect a modulation of drug metabolizing enzymes expression and function following a xenobiotic exposure. Our findings on cattle nuclear receptors, drug metabolizing enzymes and transporters gene expression and possible modulation contributed to improve the knowledge on comparative drug metabolism. Likewise, cryopreserved pig hepatocytes have been shown to be, even if stored for longer times than usual, an useful tool to study the expression and regulation of genes requiring a functional transcription factor machinery.