Disruption of the Ugt1 Locus in Mice Resembles Human Crigler-Najjar Type I Disease

Abstract

The 9 UDP-glucuronosyltranferases (UGTs) encoded by the UGT1 locus in humans are key enzymes in the metabolism of most drugs as well as endogenous substances such as bile acids, fatty acids, steroids, hormones, neurotransmitters, and bilirubin. Severe unconjugated hyperbilirubinemia in humans that suffer from Crigler-Najjar type I disease results from lesions in the UGT1A1 gene and is often fatal. To examine the physiological importance of the Ugt1 locus in mice, this locus was rendered non-functional by interrupting exon 4 to create Ugt1 ؊/؊ mice. Because UGT1A1 in humans is responsible for 100% of the conjugated bilirubin, it followed that newborn Ugt1 ؊/؊ mice developed serum levels of unconjugated bilirubin that were 40 -60 times higher than Ugt1 ؉/؊ or wild-type mice. The result of extreme unconjugated bilirubin in Ugt1 ؊/؊ mice, comparable to the induced levels noted in patients with Crigler-Najjar type 1 disease, is fatal in neonatal Ugt1 ؊/؊ mice within 2 weeks following birth. The extreme jaundice is present as a phenotype in skin color after 8 h. Neonatal Ugt1 ؊/؊ mice exhibit no detectable UGT1A-specific RNA, which corresponds to a complete absence of UGT1A proteins in liver microsomes. Conserved glucuronidation activity attributed to the Ugt1 locus can be defined in Ugt1 ؊/؊ mice, because UGT2-dependent glucuronidation activity is unaffected. Remarkably, the loss of UGT1A functionality in liver results in significant alterations in cellular metabolism as investigated through changes in gene expression. Thus, the loss of UGT1A function in Ugt1 ؊/؊ mice leads to a metabolic syndrome that can serve as a model to further investigate the toxicities associated with unconjugated bilirubin and the impact of this disease in humans.

Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

“…(ERK 1/2) in the human colon cancer cell line (HRT-18), and the genes linked to the cell cycle are significantly repressed while genes linked to the inhibition of cellular kinases are induced in Ugt-null mice (Ollinger et al, 2007;Nguyen et al, 2008).…”

“…Furthermore, many reports show that the cytotoxic effects of BR extend down to nucleus. Previous studies have demonstrated that BR induces DNA damage and disrupt cell growth via DNA oxidation and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) (Ollinger et al, 2007;Nguyen et al, 2008;Deganuto et al, 2010).…”

Life-Long Correction of Hyperbilirubinemia with a Neonatal Liver-Specific AAV-Mediated Gene Transfer in a Lethal Mouse Model of Crigler–Najjar Syndrome

“…Reactions were stopped with 50 ll of methanol with 0.02% of butylated hydroxytoluene. Samples were centrifuged at 10,000 g for 10 min at 4°C, and supernatants were collected for HPLC-MS analysis (LC-MS) as described previously (Nguyen et al, 2008) and adapted to our instrumentation.…”

“…Glucuronidation assay was performed as described previously (Nguyen et al, 2008) with minor modifications. Briefly, assays were performed after 1 hr of enzymatic reaction using the following conditions: 10 mM MgCl 2 · 6H 2 O, 50 mM TrisHCl (pH 7.7 at 37°C), 10 lg/ml phosphatidycholine, 15 lM bilirubin (bilirubin was previously dissolved in DMSO at a concentration of 0.33 lg/ll and diluted 1:10 in DMSO), 1 mM uridine diphosphate-glucuronic acid, and 1 lg/ll microsomal proteins (previously incubated for 1 hr at 4°C with digitonin at a concentration of 0.35 mg/ml of microsomes) (Gordon et al, 1984) in a total volume of 50 ll.…”

Developmental Onset of Bilirubin-induced Neurotoxicity Involves Toll-like Receptor 2-dependent Signaling in Humanized UDP-glucuronosyltransferase1 Mice

“…Knock-out of the Ugt1 locus is a lethal mutation in Ugt1 Ϫ/Ϫ mice resulting in neonatal death from severe levels of unconjugated bilirubin (5). However, expression of the human UGT1 locus in the Ugt1-null background rescues the lethality.…”