Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus

Abstract

High levels of unconjugated bilirubin (UCB) in newborn children is associated with a reduction in hepatic UDP glucuronosyltransferase (UGT) 1A1 activity that can lead to CNS toxicity, brain damage, and even death. Little is known regarding those events that lead to UCB accumulation in brain tissue, and therefore, we sought to duplicate this condition in mice. The human UGT1 locus, encoding all 9-UGT1A genes including UGT1A1, was expressed in Ugt1(-/-) mice. Because the most common clinical condition associated with jaundice in adults is Gilbert's syndrome, which is characterized by an allelic polymorphism in the UGT1A1 promoter, hyperbilirubinemia was monitored in humanized UGT1 mice that expressed either the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28))Ugt1(-/-) mice] or the normal UGT1A1*1 allele [Tg(UGT1(A1*1))Ugt1(-/-) mice]. Adult Tg(UGT1(A1*28))Ugt1(-/-) mice expressed elevated levels of total bilirubin (TB) compared with Tg(UGT1(A1*1))Ugt1(-/-) mice, confirming that the promoter polymorphism associated with the UGT1A1*28 allele contributes to hyperbilirubinemia in mice. However, TB accumulated to near toxic levels during neonatal development, a finding that is independent of the Gilbert's UGT1A1*28 promoter polymorphism. Whereas serum TB levels eventually returned to adult levels, TB clearance in neonatal mice was not associated with hepatic UGT1A1 expression. In approximately 10% of the humanized UGT1 mice, peak TB levels culminated in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures were associated with developmental milestones and can be prevented by enhancing regulation of the UGT1A1 gene in neonatal mice.

Fig. 1.

Serum TB levels in developing Ugt1 null mice and humanized UGT1 mice. At different days after birth, serum TB levels were measured from Tg(UGT1^A1*28)Ugt1^−/− mice. Triangles represent measurements taken from Ugt1^−/− mice. The circles are measurements taken from mice that showed signs of bilirubin toxicity as evident by seizure patterns (Movies S1, S2, S3, and S4). Normal ranges for TB levels in Tg(UGT1^A1*28)Ugt1^−/− mice are shown at different ages (n = 30–50 mice).

Fig. 2.

Bilirubin accumulation in the brain resulted from hyperbilirubinemia. The brain from a 7-day-old Ugt1^+/− heterozygous mouse (A) is shown compared with a Ugt1^−/− littermate (B). Also displayed are a brain from a 15-day-old Tg(UGT1^A1*28)Ugt1^−/− mouse that showed no behavioral phenotype (C) and a brain from a mouse that was developing seizures (D; Movies S1, S2, S3, and S4).

Fig. 3.

UGT1A gene and protein expression patterns in livers and small intestine from humanized UGT1 mice. (A) Liver RNA from humanized Tg(UGT1^A1*1)Ugt1^−/− and Tg(UGT1^A1*28)Ugt1^−/− liver was used in RT-PCR studies, and the isoform-specific patterns were identified in ethidium bromide-stained agarose gels. (B) Comparative values of UGT1A gene expression by RT-PCR using small-intestine RNA from Tg(UGT1^A1*1)Ugt1^−/− and Tg(UGT1^A1*28)Ugt1^−/− mice are shown. (C) The panel shows an immunoblot of microsomal protein from a pool of human liver microsomes (5 μg/lane) that were genotyped as either UGT1A1*1 (*1) or UGT1A1*28 (*28) along with liver microsomes (20 μg/lane) from Tg(UGT1^A1*1)Ugt1^−/− (*1) and Tg(UGT1^A1*28)Ugt1^−/− (*28) mice. Detection of human UGT1A1 was identified using a specific human anti-UGT1A1 antibody.

Fig. 4.

Expression of UGT1A1 in neonatal liver and small-intestine tissues from humanized UGT1 mice. (A) An immunoblot of UGT1A1 expression in liver and small intestine from Tg(UGT1^A1*28)Ugt1^−/− mice during the neonatal period is shown. (B) UGT1A1 gene-expression patterns in the liver and small intestine from Tg(UGT1^A1*1)Ugt1^−/− and Tg(UGT1^A1*28)Ugt1^−/− mice are quantitated by Q-RT-PCR at 7, 14, and 21 days after birth. The response is based on Ct values that are normalized to liver expression at 7 days. The values at 7 days are set to a 1-fold response.

Fig. 5.

Effects of phenylhydrazine treatment on serum bilirubin levels in neonatal humanized mice. Neonatal humanized UGT1 mice were administered PHZ by the i.p. route at 7 and 8 days (10 mg/kg; A), 11 and 12 days (10 mg/kg; B), 13 and 14 days (10 mg/kg; C), or 15 and 16 days (20 mg/kg; D). At 11 and 12 days of age, PHZ was also administered to wild-type mice (triangles). UCB levels were determined before the first injection and were followed by analysis every 24 h. In comparison, the escalating TB values for each treated mouse are shown by the closed circles. Open circles represent values taken from mice that progressed into seizures and died. The number of mice that escaped bilirubin-induced toxicity in each treatment group is shown. For example, in A, 13 humanized UGT1 mice were challenged with PHZ when they were 7 days old, and all of the mice eventually developed seizures resulting from the formation of kernicterus.

Fig. 6.

TCDD exposure through lactational treatment and the clearance of serum bilirubin. Maternal humanized UGT1 mice were injected by the i.p. route with 16 μg/kg TCDD shortly after giving birth. Neonatal mice were allowed to nurse freely in the cages until 14 days of age. (A) Blood TB values at 14 days are shown for the nursing mice. In comparison, the normal UCB range values recorded from our panel of untreated humanized UGT1 neonatal mice at 14 days of age are shown. (B) cDNA was synthesized from RNA collected from livers and small intestine from unexposed and TCDD-exposed 14-day-old mice. It was used for analysis of RNA content by Q-RT-PCR. The expression level of UGT1A1 was normalized by mouse CPH. (C) Expression of UGT1A1 in the liver and small intestine from microsomal preparations taken from untreated and TCDD-exposed mice was determined by Western blot analysis using an anti-human UGT1A1 antibody. (D) RNA from livers and small intestine was used in Q-RT-PCR experiments to measure Cyp1a1 gene expression.