Porcine model of xenobiotic metabolism

A xenobiotic is a foreign chemical substance found in the environment. The body removes xenobiotics by xenobiotic metabolism. Drug metabolizing enzymes (DMEs) play central roles in the metabolism, elimination, and detoxification of xenobiotics introduced into the body. Orphan nuclear receptors play crucial role in regulation of the expression of DMEs. The pig has quickly grown into an important biomedical research tool over the past few decades. The pig is an appropriate animal model for the investigation of xenobiotic disposition, as the transporters and CYP enzymes are very similar to those in humans. The characterization of porcine drug metabolism genes and the genes involved in regulating drug metabolism can provide insights into human drug metabolic diseases and individual variability of responses toward a drug. The tissue- and stage-specific expression of the nuclear receptors in pigs and their comparison to humans will be of great interest. Consequently, the goal of the proposal is to validate pig as a model of xenobiotic metabolism in order to get a better understanding of the pharmacokinetic properties of the xenobiotics. Expression of orphan nuclear receptors were screened across various porcine organs (liver, kidney, lung, small intestine, spleen, pancreas, heart, brain and skeletal muscle). Analysis of the mRNA expression levels of porcine orphan nuclear receptors in total RNA from various porcine organs was also performed by real time reverse transcriptase PCR. Expression of all the porcine nuclear receptors studied except (PPARγ) was detected in the liver and kidney. Most of the nuclear receptors showed higher expression in the liver. The tissue distribution and the expression profiles of the porcine nuclear receptors were consistent with those of human. To evaluate the effect of xenobiotic exposure on the expression pattern of the nuclear receptors, expression pattern of nuclear receptors were evaluated in three different developmental stages i.e; three month old fetus, one month old piglet and one year old adult pig. The expression levels of the nuclear receptors in adult tissues were higher than that of one month old piglets which in turn were higher than those of a three month old fetal piglet. Porcine orphan nuclear receptors liver X receptor alpha (LXRα), liver X receptor beta (LXRβ) and constitutive androstane receptor (CAR) were cloned and the sequence analysis revealed eight novel transcript variants for LXRα and LXRβ each and five novel transcript variants for CAR. The expression profiles and the physiochemical properties of the novel identified transcript variants were analyzed. Further, we developed and characterized a porcine hepatocyte cell line representative of human primary hepatocytes to support drug toxicity and metabolism assessments. Three independent hepatocyte cell lines were developed from three different Oncopigs and all of them expressed hepatocyte specific and most important drug metabolism and regulation genes comparable to those porcine primary hepatocytes. We evaluated the effect of selective CYP modulators on three porcine hepatocyte cell lines. All the three independent porcine hepatocyte cell lines behaved the same way and the gene regulation pattern in hepatocyte cell lines was similar to that of primary hepatocytes and human models. These findings indicate that this porcine hepatocyte cell line represents a useful and predictive model for high throughput screening of new drugs as well as studies on metabolism and hepatotoxicity of chemicals