UDP-glucuronosyltransferase activity in human liver and colon Christian P. Strassburg, Nghia Nguyen, Michael P. Manns, Robert H. Tukey  Gastroenterology  Volume 116, Issue 1, Pages 149-160 (January 1999) DOI: 10.1016/S0016-5085(99)70239-8 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Graphic representation of the 54 specific activities determined for colon (mean, 5 samples) and liver (mean, 5 samples) microsomal protein. (A) Substrates with high activities and differences between liver and colon microsomal UGT activity. (B) Lower-activity substrates analyzed with hepatic and colonic microsomal protein. (C) Substrates identified to comparable specific activities in human colon and liver. Alpha-NF, α-naphthoflavone; beta-NF, β-naphthoflavone; alpha-NA, α-naphthylamine; 1,8-dOH-AQ, 1,8-dihydroxy anthraquinone; pOH-BA, parahydroxybenzoic acid; cis-RA, cis retinoic acid; all-trans-RA, all-trans retinoic acid; 4,2-dOH-DPHA, 1,2-dihydroxy diphenylamine; 2-OH-estradiol, 2-hydroxy estradiol; 2-OH-estriol, 2-hydroxy estriol; 17-OH-PG, 17-hydroxy progesterone; 4-OHestrone, 4-hydroxy estrone; 4-OH-coumarin, 4-hydroxy coumarin; 8-OH BAP, 8-hydroxy benzo(α)pyrene; 2-OH-biphenyl, 2-hydroxy biphenyl; tert-butylphenol, 4-tert-butylphenol; 4-OH-biphenyl, 4-hydroxy biphenyl; 4-MU, 4 methylumbelliferone; HDCA, hyodeoxycholic acid; 7OH-flavone, 7-hydroxy flavone; 7-OH-BAP, 7-hydroxy benzo(α)pyrene. Bars represent standard deviations. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Graphic representation of the 54 specific activities determined for colon (mean, 5 samples) and liver (mean, 5 samples) microsomal protein. (A) Substrates with high activities and differences between liver and colon microsomal UGT activity. (B) Lower-activity substrates analyzed with hepatic and colonic microsomal protein. (C) Substrates identified to comparable specific activities in human colon and liver. Alpha-NF, α-naphthoflavone; beta-NF, β-naphthoflavone; alpha-NA, α-naphthylamine; 1,8-dOH-AQ, 1,8-dihydroxy anthraquinone; pOH-BA, parahydroxybenzoic acid; cis-RA, cis retinoic acid; all-trans-RA, all-trans retinoic acid; 4,2-dOH-DPHA, 1,2-dihydroxy diphenylamine; 2-OH-estradiol, 2-hydroxy estradiol; 2-OH-estriol, 2-hydroxy estriol; 17-OH-PG, 17-hydroxy progesterone; 4-OHestrone, 4-hydroxy estrone; 4-OH-coumarin, 4-hydroxy coumarin; 8-OH BAP, 8-hydroxy benzo(α)pyrene; 2-OH-biphenyl, 2-hydroxy biphenyl; tert-butylphenol, 4-tert-butylphenol; 4-OH-biphenyl, 4-hydroxy biphenyl; 4-MU, 4 methylumbelliferone; HDCA, hyodeoxycholic acid; 7OH-flavone, 7-hydroxy flavone; 7-OH-BAP, 7-hydroxy benzo(α)pyrene. Bars represent standard deviations. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Graphic representation of the 54 specific activities determined for colon (mean, 5 samples) and liver (mean, 5 samples) microsomal protein. (A) Substrates with high activities and differences between liver and colon microsomal UGT activity. (B) Lower-activity substrates analyzed with hepatic and colonic microsomal protein. (C) Substrates identified to comparable specific activities in human colon and liver. Alpha-NF, α-naphthoflavone; beta-NF, β-naphthoflavone; alpha-NA, α-naphthylamine; 1,8-dOH-AQ, 1,8-dihydroxy anthraquinone; pOH-BA, parahydroxybenzoic acid; cis-RA, cis retinoic acid; all-trans-RA, all-trans retinoic acid; 4,2-dOH-DPHA, 1,2-dihydroxy diphenylamine; 2-OH-estradiol, 2-hydroxy estradiol; 2-OH-estriol, 2-hydroxy estriol; 17-OH-PG, 17-hydroxy progesterone; 4-OHestrone, 4-hydroxy estrone; 4-OH-coumarin, 4-hydroxy coumarin; 8-OH BAP, 8-hydroxy benzo(α)pyrene; 2-OH-biphenyl, 2-hydroxy biphenyl; tert-butylphenol, 4-tert-butylphenol; 4-OH-biphenyl, 4-hydroxy biphenyl; 4-MU, 4 methylumbelliferone; HDCA, hyodeoxycholic acid; 7OH-flavone, 7-hydroxy flavone; 7-OH-BAP, 7-hydroxy benzo(α)pyrene. Bars represent standard deviations. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Autoradiography of a thin-layer liquid chromatography plate showing the separation of androsterone glucuronides catalyzed by 25 μg of liver (left) and colonic (right) microsomal protein in each lane. Specific catalytic activities in all 5 colon samples are lower than in the liver samples (numbering corresponds to Figure 1 and Tables 1 and 2). Arrow indicates direction of thin-layer chromatography separation. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Specificity of the rabbit anti-human UGT1A antibody. A Western blot using recombinant UGT1A1, UGT1A4, UGT1A7, and UGT2B15 protein demonstrated selective binding to UGT1A proteins (top). The same proteins, immunodetected with an antibody specific for both UGT1A and UGT2B protein, demonstrated the presence of all used proteins. Standard, molecular-weight marker; Sf9, Spodoptera frugiperda wild-type protein; anti-UGT1A, rabbit anti-human UGT1A antibody2; anti-UGT, sheep anti-rabbit UGT antibody.23 Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Indirect immunofluorescence analysis of human (A and B) liver tissue sample 4 and (C–E) colon sample 1 showing UGT1A expression in hepatocytes and colon epithelial cells. Rabbit anti-human UGT1A antibody directed against a 15-mer of the UGT1A common exon 5 was diluted 1:160 and used for A, C, and E. In B and D normal rabbit serum was used as a negative control. (A) In liver homogeneous staining was observed throughout the lobulus (bile duct shown by arrowhead) and was most pronounced around the nucleus of each cell. (B) The negative control showed minor unspecific staining of the portal field. (C) In colon staining was confined to the epithelial layer and did not include the lamina propria or submucosa (arrowhead). Longitudinal (arrow) and transverse sections of the mucosal crypts are visible. (D) A control serum failed to stain colon tissue. (E) An enlarged section from C shows staining of epithelial colon cells excluding the nuclei, as also seen in liver (arrow). Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Overall UGT1A mRNA levels detected by quantitative DRT-PCR in 5 liver (■) and 5 colon (▴) RNA samples; (B) overall UGT1A protein detected by Western blot in 25-μg aliquots of 4 liver and 4 colon microsomal protein samples. UGT1A mRNA concentration was higher in all 5 liver samples than in the colon samples with differences not exceeding 50%. UGT1A protein was found to be expressed at comparable levels in both tissues. Note banding pattern differences between colon and liver microsomes, suggesting the expression of different UGT1A protein species. rUGT1A1, recombinant UGT1A1 expressed in baculovirus, separated as a control. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Overall UGT1A mRNA levels detected by quantitative DRT-PCR in 5 liver (■) and 5 colon (▴) RNA samples; (B) overall UGT1A protein detected by Western blot in 25-μg aliquots of 4 liver and 4 colon microsomal protein samples. UGT1A mRNA concentration was higher in all 5 liver samples than in the colon samples with differences not exceeding 50%. UGT1A protein was found to be expressed at comparable levels in both tissues. Note banding pattern differences between colon and liver microsomes, suggesting the expression of different UGT1A protein species. rUGT1A1, recombinant UGT1A1 expressed in baculovirus, separated as a control. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Tissue-specific expression of UGT1A isoform mRNAs in the liver and colon samples detected by DRT-PCR. All 5 liver samples expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A9, whereas all 5 colon samples additionally expressed UGT1A8 and UGT1A10, which is in agreement with previously published findings.2 Significant differences of UGT1A-specific RNA isoforms between the different samples were not observed. Standard, HaeIII digest of φX174 DNA. Gastroenterology 1999 116, 149-160DOI: (10.1016/S0016-5085(99)70239-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions