nitrophenols and Crigler-Najjar-Syndrome

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bilirubin; Camptothecin; Causality; Crigler-Najjar Syndrome; Glucuronosyltransferase; Guanine; Humans; Irinotecan; Isoenzymes; Microsomes, Liver; Nitrophenols; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Uridine Diphosphate; Zidovudine

In vitro glucuronidation was studied in liver microsomes from two patients with Crigler-Najjar type I (CN-I) disease and compared with the activity measured in microsomes prepared from six control human livers. The UDP-glucuronosyltransferase (UGT) activity was determined toward the following substrates: 4-nitrophenol, propofol, (-)-morphine (formation of the 3-glucuronide), and diflunisal (formation of the phenolic and acyl glucuronides). Glucuronidation of 4-nitrophenol was reduced in one of the CN-I livers (CN-I No. 1) (0.9 nmol min(-1)mg(-1)) and normal in the other CN-I liver (CN-I No. 2) (3.5 nmol min(-1) mg(-l)) compared to the control livers (5.6 +/- 29 nmol min(-1) mg(-1)), mean +/- S.D.). Propofol glucuronidation was not detectable (i.e. less than 0.100 nmol min(-l) mg(-1) in the CN-I No. 1 liver and normal in the CN-I No. 2 liver (1.78 nmol min(-1) mg(-1) against 1.52 +/ 0.72 nmol min(-l) mg(-) in the control livers). The glucuronidation of (-)-morphine to the 3-glucuronide and the formation of the phenolic and acyl glucuronides of diflunisal were normal in both CN-I livers compared to the control livers. Our results show that CN-I patients are heterogeneous regarding UGT activity toward the phenolic substances 4-nitrophenol and propofol.

Topics: Analgesics, Opioid; Anesthetics, Intravenous; Anti-Inflammatory Agents, Non-Steroidal; Child, Preschool; Crigler-Najjar Syndrome; Cytochrome P-450 Enzyme System; Diflunisal; Female; Glucuronates; Glucuronosyltransferase; Humans; In Vitro Techniques; Male; Microsomes, Liver; Morphine; Nitrophenols; Propofol

Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.

Topics: Androsterone; Base Sequence; Bilirubin; Blotting, Northern; Chromosome Mapping; Crigler-Najjar Syndrome; Electrophoresis, Agar Gel; Estradiol; Glucuronosyltransferase; Humans; Hymecromone; Isoenzymes; Liver; Molecular Sequence Data; Nitrophenols; Polymerase Chain Reaction; RNA, Messenger