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Bile salt hydroxylation as a mechanism for detoxification in SPGP knockout mice Forrest, Dana Nichole

Abstract

In humans, mutations in the Sister of P-glycoprotein (spgp) gene involved in bile salt transport lead to progressive familial intrahepatic cholestasis type 2 (PFIC2). Generation of the spgp[sup -/-] mice demonstrated that the mice are not affected as severely as humans. Analysis of the biliary bile salt pool of the spgp[sup -/-] mice indicated the presence of tetrahydroxylated bile salts. However, the transport rate of the hydrophilic bile salts was unaffected. Examination of tetrahydroxylated bile salts as a possible detoxification mechanism requires identification and characterization of the enzyme involved. Development of an HPLC bile salt separation method generated bile salt profiles of spgp[sup -/-] and wild-type mice fed a normal or 0.5% cholic acid (CA) enriched diet. Analysis of the profiles suggested the 2-day change in diet had little effect on the already elevated levels of tetrahydroxylated bile salt levels in the spgp[sup -/-] mice. However, wild-type mice produced increased levels of the tetrahydroxylated bile salt when fed the 0.5% CA diet. Three tetrahydroxylated bile salt species were present in the HPLC profiles. Expression analysis of the CYP3A family, the three major enzymes in bile salt synthesis, and five of the main nuclear receptors involved in the regulation of cholesterol and bile salt metabolism was performed using Northern Blot analysis and real-time PCR. When tetrahydroxylated bile salt levels were compared to the mRNA expression levels, there was little correlation to the candidate genes tested. Furthermore, the reduction in CYP7A1 and CYP8B1 expression levels under the 0.5% CA supplemented diet indicated substrates from these pathways are not involved in the synthesis of the tetrahydroxylated bile salts. Consequently, it is believed that bile salt tetrahydroxylation is either regulated at the protein level or is due to expression of a gene not tested.

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