Developmental aspects of human hepatic drug glucuronidation in young children and adults

Abstract

Background and aims: The liver represents one of the major sites of human glucuronidation. Many therapeutic drugs are substrates for UDP-glucuronosyltransferases (UGT) leading to the formation of usually inactive glucuronides. Hepatic glucuronidation undergoes significant changes during fetal and neonatal development requiring age adapted drug therapy. Regulation of individual UGT genes during hepatic development has not been defined.

Subjects and methods: Expression of 13 UGT genes and glucuronidation activities were analysed in 16 paediatric liver samples (aged 7-24 months), two fetal samples, and 12 adult liver samples (aged 25-75 years) using duplex reverse transcription-polymerase chain reaction, western blot, and specific catalytic UGT activity assays.

Results: No UGT transcripts were detected in fetal liver at 20 weeks' gestation. In contrast, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, and UGT2B15 transcripts were present without variation in all 28 hepatic samples after six months of age. Significantly lower expression of UGT1A9 and UGT2B4 mRNA was identified in paediatric liver. Hepatic glucuronidation activity in children aged 13-24 months was found to be lower than in adults for ibuprofen (24-fold), amitriptyline (16-fold), 4-tert-butylphenol (40-fold), estrone (15-fold), and buprenorphine (12-fold).

Conclusions: An early phase characterised by the appearance of UGT gene transcripts and a later phase characterised by upregulation of UGT expression is demonstrated during human hepatic development. The differential regulation of UGT1A9 and UGT2B4 expression extends beyond two years of age and is capable of influencing hepatic glucuronidation of common therapeutic drugs in children. The development of hepatic UGT activities is significant for paediatric drug therapy and the prevention of adverse drug effects.

Figure 1

Expression of 13 UGT1A and UGT2B transcripts detected by exon specific duplex reverse transcription-polymerase chain reaction. The ethidium bromide stained gel shows representative examples of fetal liver (20 weeks' gestation, male), child liver at nine months (male), and an example of adult liver (45 years, male). De novo regulation of the UGT1A gene locus and the four UGT2B genes is apparent between the fetal and child liver samples. Between the child and adult livers the differential upregulation of UGT transcripts is visible.

Figure 2

Significant differential downregulation of UGT1A9 and UGT2B4 mRNA in child liver. Graphic representation of the quantification of mRNA levels analysed by duplex reverse transcription-polymerase chain reaction and calculated relative to the presence of β-actin. Statistical analysis demonstrated significant differential regulation only of UGT1A9 and UGT2B4 mRNA in children 6–18 months of age. This finding demonstrates the independent development of UGT isoform expression in human liver. ***p<0.001.

Figure 3

Expression of UGT1A1, UGT1A6, and UGT2B7 protein during hepatic development. Western blot analysis demonstrating the specific detection of UGT1A1, UGT1A6, and UGT2B7 protein in seven child and adult liver samples. As demonstrated in the duplex reverse transcription-polymerase chain reaction analysis of UGT1A1, UGT1A6, and UGT2B7 transcript expression (compare with fig 2 ▶), no significant differences were detected at the protein level, confirming the transcript analysis. All samples were found to express immunodetectable UGT protein indicating the high quality of the samples. Neg control (Sf9), Spodoptera frugiperda cells not expressing UGT protein used for negative control experiments; Pos control, positive control using the corresponding recombinant UGT for the employed antibody.

Figure 4

Differences in catalytic activity between adult and child liver. Graphic representation of the analysis of 18 substrates tested for glucuronidation using child and adult liver. 2-OH-biphenyl, 2-hydroxybiphenyl; 4-OH-coumarin, 4-hydroxycoumarin; 2-OH-estrone, 2-hydroxyestrone; 4-OH-estrone, 4-hydroxyestrone.