Superfund Research Program

Antioxidant Protection by Hyperbilirubinemia in Toxicity and Disease

Project Leader: Robert H. Tukey
Grant Number: P42ES010337
Funding Period: 2000-2017

Project-Specific Links

Final Progress Reports

Year:   2016  2009  2004 

The development of humanized animal models that express human xenobiotic metabolizing genes and proteins has been the focus of this laboratory. By introducing entire human genes into mice through transgenic technology coupled with the knockout of murine homologous genes it is possible to examine the influence of humoral factors on gene control contributed by tissue specific control, hormonal influences and induction by xenobiotic or environmental toxicant exposure. Unlike conventional in vitro tools such as tissue culture, the development of humanized mice provides the unique opportunity to allow the global influences from homeostatic balance to be examined through gene expression, protein function and the possible effects of human polymorphisms on xenobiotic induced phenotypes. Environmental induced toxicities are dependent upon the steady-state balance between biologically active toxicant and the percentage or rate of detoxification. One of the key detoxification processes in all cells and tissues is the conversion of dangerous toxicants into harmless water soluble compounds by the addition of glucuronic acid by the Superfamily of UDP-glucuronosyltransferases (UGTs). Although there are two gene families, each consisting of 9 unique UGTs, the UGT1 family encodes the key proteins linked to toxicant metabolism. In addition, all 9-,UGT1A genes are encoded by the unique UGT1 locus, which spans over 200 kb on chromosome 2. To examine the functionality of these proteins and to link their importance to toxicant metabolism and clearance, humanized UGT1 mouse lines were developed.

In 2008, the laboratory generated the first knockout of a Ugt gene by interrupting exon 4 of the mouse Ugt1 locus (Nguyen, et al. JBC 283:7901, 2008). Because Ugt1a1 and the other 9-Ugt1a genes were non-functional, this mutation led to neonatal lethality at approximately 7 days, resulting from the inability to glucuronidate serum bilirubin. Using heterozygous Ugt1^+/- mice, the researchers crossed these mice with our transgenic UGT1 mouse line, which carries and expresses the entire human UGT1 locus. The result of this cross followed by back crosses led to mice that were humanized for the UGT1 locus in a Ugt1-null background. Humanized UGT1 mice recovered the neonatal lethality. In addition, the UGT1 locus in these animals encodes the Gilbert’s UGT1A1*28 allele. In humans, Gilbert’s syndrome results in benign hyperbilirubinemia because of a promoter polymorphism in the UGT1A1 gene. To our surprise, humanized UGT1 mice were also hyperbilirubinemic because of limited expression of liver UGT1A1. When researchers employed these mice to examine their ability to metabolize important clinical therapeutics such as the cancer drug SN-38, the levels of metabolism and clearance were dramatically inhibited because of limited hepatic UGT1A1 expression. In addition, they demonstrated that the popular anti-hypolipidemic drug ezetimibe (Zetia) was not a substrate for UGT1A1, as previously thought, an important finding for clinical and toxicity considerations when given to Gilbert’s patients.

Since high levels of unconjugated bilirubin (UCB) are often seen in young children, the researchers analyzed the levels of UCB in newborn/neonatal humanized UGT1 mice. This was particularly relevant since toxic levels of UCB in humans can develop, leading to CNS toxicity, brain damage and even death. In humanized UGT1 mice, UCB accumulates to near toxic levels during neonatal development. While serum UCB levels eventually return to adult levels, UCB clearance in neonatal mice is not associated with hepatic UGT1A1 expression. Importantly, in approximately 10% of the humanized mice, peak UCB levels culminate in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures are associated with developmental mile stones and can be prevented by enhancing regulation of the human UGT1A1 gene in neonatal mice. The human UGT1A1 gene plays a key role in the metabolism and disposition of many environmental toxicants, since it is regulated by virtually all of the xenobiotic and nuclear receptors. Because of its potential to undergo regulation in vivo and the ease of measuring serum UCB as a biomarker of control, these mice will serve to identify selective toxicants that target the different transcriptional modifies which regulate the UGT1A1 gene.