The Expression Of UGT1A9 From Human Liver Tissues In CHL Cell Lines And The Study Of Glucuronidation Function To Drugs

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum that catalyze the conjugation of a broad variety of endogenous and exogenous lipophilic aglycon substrates (such as bilirubin, steroid hormone and drugs, insecticide, etc) with glucuronic acid using UDP-glucuronic acid (UDPGA) as the sugar donor. UGTs are a gene superfamily of phase II drug-metabolizing enzymes, they are responsible for the glucuronidation of a significant number of different functional groups (e.g. -OH> -COOH-. -NH^ -SH). Glucuronidation is a major factor in the elimination of lipophilic compounds from the body, and is the most important pathway in phase II metabolism.UGT1A9 is a member of UGT1A subfamily. The gene of UGT1 family is constituted of four common exons and one specific first exon. There are at least 12 kinds of the first exon identified in human being, among which nine exons belong to UGT1A subfamily. No gene polymorphism about UGT1A9 is reported.Human UGTs are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues. Liver is the main organ for drug metabolism, it nearly have all UGT isozymes. But some of UGTs can only be found in limited organs. UGT1A9 is mainly existed in liver, but some reports indicated that the expression of UGT1A9 were also found in some extrahepatic tissues such as colon, kidney, prostate, testis, breast, ovary, skins and some tumor tissues.UGT1A9 can be induced by polycyclic aromatic hydrocarbons (PAHs)and other compounds such as antioxident t-butylhydroquinone. There were limited reports about the metabolism catalyzed by UGT1A9. UGT1A9 mainly catalyze the glucuronidationof hydroxyl to produce O-glucuronide. Besides, UGT1A9 also catalyze the glucuronidation of carboxyl and hydroxylamine compounds. No report was found about the metabolism of isoflavone by UGT1A9.The substrates metabolized by UGTs encompass a wide range of structurally unrelated substances. Previous studies revealed that the individual UGTs isozymes tend to exhibit un-strict and overlapping patterns of substrate specificity.Knowledge of distribution and regulation of UGTs remains limited. Liver microsomes is a frequently used enzyme source in in vitro study of glucuronidation, but it has the following deficient respects:1. Liver microsome is a mixture of various enzymes, and therefore the results from microsome cannot represent the function of a individual isozyme.2. The substrate spectrum of UGTs has obvious difference between species and individual.3. Some of UGTs isozyme expressed in other tissues instead of liver.Now transgenic cell lines that express the protein encoded by UGT gene were broadly used instead of liver microsomes. The enzyme expressed in cell lines helps to realize the function of a specific gene and will provide direct information related to the isozyme interested.Glucuronidation catalyzed by UGTs have been a subject of intense research during the last decades, but no domestic literature was found about the investigation in this area, and the establishment of UGTs transgenic cell lines is not reported either. This thesis is the first domestic article in China that reports the cloning of UGT1A9 from a Chinese liver tissue and expression in cell lines. The expression vector pREP9 was employed for the effective expression of UGT1A9 in Chinese hamster lung cells (CHL). The cell lines established were used for the study of glucuronidation in vitro, and the drug metabolism characteristics were summarized.As has been indicated that UGT1A9 mainly glucuronide hydroxyl, compounds that have aliphatic or phenol hydroxyl were selected for substrates of UGT1A9. Propofol and paracetamol are compounds that have phenol hydroxyl and propranolol and propafenone are those that have aliphatic ones. Among the four compounds, propofol is the specific substrate of UGT1A9 and can be served as the comparison reference of enzymatic activity. Based on the study of four compound, four kinds of isoflavone compounds, i.e., quercetin, kaempferol,...