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Ontogeny of hepatic drug metabolizing enzymes in sheep Pretheeban, Manoja

Abstract

Administration of therapeutic, supplemental and social drugs to the mother during pregnancy exposes the fetus to these medicines and their metabolites, which can cause defects in nervous, renal, cardiac and metabolic functions. Hepatic metabolic enzymes such as cytochrome P450 (CYP) 2A6, 2C19, 2D6 and UDP glucuronosyltransferases (UGT) 1A6, 1A9, 2B7 are important for the metabolism of many drugs. In order to understand whether these drugs are safe enough during pregnancy and in postnatal period, knowledge of the expression levels of these enzymes in different developmental stages is important. Therefore we proposed to clone the CYP and UGT genes and quantify these enzymes at gene and protein levels at different developmental stages such as fetus, newborn and adults. This study was performed in sheep (adult n=4; newborn n=3; and fetus n=3) liver to compare the mRNA and protein expression levels of the above enzymes and a regulatory factor, Hepatic Nuclear Factor 4α (HNF4α). The effect of antenatal glucocorticoid on these enzymes was also studied by infusion of cortisol (0.45mg/h; 80h) to another group of fetuses (n=5). Sheep sequences were cloned and real time PCR was performed to analyze the relative mRNA expression levels in the above four groups. Microsomes were prepared and western blot analysis was performed using human or rat antibodies to measure the relative protein expression levels. In terms of mRNA and protein expression of the above mentioned enzymes, fetal and newborn levels were very low compared to the adult. Some CYP proteins (CYP2A, CYP2C) were absent in the fetus and even in newborns (CYP2A). Sheep UGT protein levels were not measured since the human UGT antibodies did not work. Glucocorticoid plays a role in up regulating both the mRNA and protein expression of CYP2D6. Moreover the correlation observed between the above enzymes and HNF4α indicates a possible regulatory role in sheep similar to that in humans. The findings of this study follow a similar pattern found in the human and indicate that fetal and newborn lambs have a reduced ability to metabolize drugs that are substrates of these CYP isoforms.

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