Human UGT1A8 and UGT1A10 mRNA are expressed in primary human hepatocytes

Abstract

It is widely believed that the UGT1A isoforms, UGT1A8 and -1A10, are expressed exclusively in extrahepatic tissues. In this work, human primary hepatocytes from six donors were analyzed for UGT1A8 and -1A10 mRNA expression by semi-quantitative RT-PCR. New primers to amplify UGT1A8 mRNA were designed and found to differ from those previously published. We demonstrated that UGT1A8 and -1A10 mRNA are expressed in hepatocytes. Although basal UGT mRNA levels were detected in untreated hepatocytes, significant up-regulation of the levels of mRNA for these isoforms were seen after treatment with 3-methylcholanthrene (3-MC) and rifampicin (Rif). RT-PCR products for all UGTs were sequenced and unambiguously identified as matching the corresponding cDNA. The discovery of these isoforms in hepatocytes is a novel discovery and will stimulate studies on the potential role for these isoforms in hepatic detoxification.

Fig. 1. Localization of primers and RT-PCR products

Isoform-specific sense primers are located within the first exon. The different anti-sense primers used are located within the common 3′ sequence (exons 2–5) so as to amplify across the exon boundaries in mRNA from the target genes.

Fig. 2. UGT1A8, –1A9, and –1A10 mRNA levels in primary human hepatocytes from 6 donors

Upon receipt of hepatocytes in 6-well culture plates, plating medium was replaced with fresh HMM supplemented with 0.1 μM insulin, 0.1 μM dexamethasone, 50 μg/mL gentamicin, and 0.25 μg/mL amphotericin B (designed HMM⁺). After an overnight equilibration period at 37°C under an atmosphere of 95% air/5% CO₂, hepatocytes were harvested and total RNA was prepared with Trizol. UGT1A8, UGT1A9 and UGT1A10 mRNA levels expression was analyzed by semi-quantitative RT-PCR (described under “Experimental Procedures”). Data are the mean±SD of four data points.

Fig. 3. 3-MC and Rif increase UGT1A8 and -1A10 levels in primary human hepatocytes from donor HH1117

Upon receipt of hepatocytes in 6-well culture plates, plating medium was replaced with fresh HMM⁺. After an overnight equilibration period at 37°C under an atmosphere of 95% air/5% CO₂, hepatocytes were treated with 3-MC (1 μM) or Rif (15 μM) for 24 hr. UGT1A8 and –1A10 mRNA expression was analyzed by semi-quantitative RT-PCR. Results are normalized to fold of control (treated cells versus vehicle), and data are the mean±SD of four data points.

Fig. 4

Comparison of RT-PCR products generated using mRNA from 6 sets of hepatocytes and the UGT1A8-G and UGT1A8-C primers. PCR and incubation conditions are as in Figs. 2 and 3. Expression of GAPDH is included as a control.

Fig. 5

Alignment of RT-PCR products generated by UGT1A8G and –1A8C primers with UGT1A8*1 (A¹⁷³C²⁷⁷) (AF462268), –1A8*2 (G¹⁷³C²⁷⁷) (AF462267), and –1A8*3 (A¹⁷³Y²⁷⁷) (AF465200).

Fig. 6. RT-PCR analysis of mRNA expression of UGT1A10, and –1A8 in the human GI tract

RT-PCR was carried out as described in the text using the UGT1A8-G and –1A8-C primers and freshly prepared human intestinal mucosa (S1–S4, segments of remaining small intestine; C: colon) from Donor 1 (43 year-old male; cause of death: overdose of Atavan and valproic acid; history: valproic acid, Zyprexa, Haladol, Atavan) and Donor 2 (39 year-old male; cause of death: motor vehicle accident). The bar graphs show the results of quantitation by densitometry of ethidium bromide gel electrophoresis of the RT-PCR amplicons obtained using UGT1A10, –1A8-C, 1A8-G, and GAPDH (shown below each graph) primers. The results for each amplicon have been normalized to the recovery of GAPDH mRNA.