Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase

Jurgen Seppen, Kouji Tada, Sandra Hellwig, Conny T M Bakker, Vinayaka R. Prasad, Namita Roy Chowdhury, Jayanta Roy-Chowdhury, Piter J. Bosma, Ronald P J Oude Elferink

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Abstract

Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in bilirubin metabolism of CN patients is the absence of B-UGT activity. The transplantation of cells able to glucuronidate bilirubin should therefore lower serum bilirubin levels. The Gunn rat is the animal model of CN disease. Primary Gunn rat fibroblasts (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA. A cell line was obtained expressing B-UGT at a level comparable to hepatocytes. Bilirubin added to the culture medium of these cells was glucuronidated and excreted. The B-UGT activities of transduced GURF and freshly isolated Wistar hepatocytes were compared at different bilirubin concentrations. The specific B-UGT activities of these two cell types were comparable when physiological bilirubin concentrations (5-10 μM) were present in the culture media. At higher bilirubin concentrations (20-80 μM) the hepatocytes were more active than the transduced GURF. We conclude that with the addition of only one enzyme (B-UGT) fibroblasts can perform the complete set of reactions necessary for bilirubin glucuronidation. The difference in B-UGT activity between transduced GURF and hepatocytes at 20-80 μM bilirubin can be explained by lower UDP-glucuronic acid and glutathione S-transferase levels in GURF. Our findings also indicate that these cells could be used to develop extrahepatic gene therapy for CN disease.

Original languageEnglish (US)
Pages (from-to)477-483
Number of pages7
JournalBiochemical Journal
Volume314
Issue number2
StatePublished - Mar 1 1996

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bilirubin glucuronoside glucuronosyltransferase
Gunn Rats
Fibroblasts
Bilirubin
Rats
Hepatocytes
Metabolism
Culture Media
Uridine Diphosphate Glucuronic Acid
Gene therapy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Seppen, J., Tada, K., Hellwig, S., Bakker, C. T. M., Prasad, V. R., Roy Chowdhury, N., ... Oude Elferink, R. P. J. (1996). Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase. Biochemical Journal, 314(2), 477-483.

Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase. / Seppen, Jurgen; Tada, Kouji; Hellwig, Sandra; Bakker, Conny T M; Prasad, Vinayaka R.; Roy Chowdhury, Namita; Roy-Chowdhury, Jayanta; Bosma, Piter J.; Oude Elferink, Ronald P J.

In: Biochemical Journal, Vol. 314, No. 2, 01.03.1996, p. 477-483.

Research output: Contribution to journalArticle

Seppen, J, Tada, K, Hellwig, S, Bakker, CTM, Prasad, VR, Roy Chowdhury, N, Roy-Chowdhury, J, Bosma, PJ & Oude Elferink, RPJ 1996, 'Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase', Biochemical Journal, vol. 314, no. 2, pp. 477-483.
Seppen J, Tada K, Hellwig S, Bakker CTM, Prasad VR, Roy Chowdhury N et al. Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase. Biochemical Journal. 1996 Mar 1;314(2):477-483.
Seppen, Jurgen ; Tada, Kouji ; Hellwig, Sandra ; Bakker, Conny T M ; Prasad, Vinayaka R. ; Roy Chowdhury, Namita ; Roy-Chowdhury, Jayanta ; Bosma, Piter J. ; Oude Elferink, Ronald P J. / Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase. In: Biochemical Journal. 1996 ; Vol. 314, No. 2. pp. 477-483.
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abstract = "Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in bilirubin metabolism of CN patients is the absence of B-UGT activity. The transplantation of cells able to glucuronidate bilirubin should therefore lower serum bilirubin levels. The Gunn rat is the animal model of CN disease. Primary Gunn rat fibroblasts (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA. A cell line was obtained expressing B-UGT at a level comparable to hepatocytes. Bilirubin added to the culture medium of these cells was glucuronidated and excreted. The B-UGT activities of transduced GURF and freshly isolated Wistar hepatocytes were compared at different bilirubin concentrations. The specific B-UGT activities of these two cell types were comparable when physiological bilirubin concentrations (5-10 μM) were present in the culture media. At higher bilirubin concentrations (20-80 μM) the hepatocytes were more active than the transduced GURF. We conclude that with the addition of only one enzyme (B-UGT) fibroblasts can perform the complete set of reactions necessary for bilirubin glucuronidation. The difference in B-UGT activity between transduced GURF and hepatocytes at 20-80 μM bilirubin can be explained by lower UDP-glucuronic acid and glutathione S-transferase levels in GURF. Our findings also indicate that these cells could be used to develop extrahepatic gene therapy for CN disease.",
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AU - Prasad, Vinayaka R.

AU - Roy Chowdhury, Namita

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