Cytosolic sulfotransferases (SULTs) are expressed during early life and therefore metabolize endogenous and xenobiotic chemicals during development. Little is currently known about the regulation of individual SULTs in the developing human liver. We characterized SULT expression in primary cultures of human fetal hepatocytes and the HepaRG model of liver cell differentiation. SULT1A1 (transcript variants 1-4), SULT1C2, SULT1C4, SULT1E1, and SULT2A1 were the most abundant transcripts in human fetal hepatocytes. In HepaRG cells, SULT1B1, SULT1C2/3/4, and SULT1E1 mRNA levels increased during the transition from proliferation to confluency and then decreased as the cells underwent further differentiation. By contrast, SULT2A1 mRNA levels increased during differentiation, whereas SULT1A1 and SULT2B1 mRNA levels remained relatively constant. The temporal patterns of SULT1C2, SULT1E1, and SULT2A1 protein content were consistent with those observed at the mRNA level. To identify regulators of SULT expression, cultured fetal hepatocytes and HepaRG cells were treated with a panel of lipid- and xenobiotic-sensing receptor activators. The following effects were observed in both fetal hepatocytes and HepaRG cells: 1) liver X receptor activator treatment increased SULT1A1 transcript variant 5 levels; 2) vitamin D receptor activator treatment increased SULT1C2 and SULT2B1 mRNA levels; and 3) farnesoid X receptor activator treatment decreased SULT2A1 expression. Activators of aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, and peroxisome proliferator-activated receptors produced additional gene-dependent effects on SULT expression in HepaRG cells. These findings suggest that SULT-regulating chemicals have the potential to modulate physiologic processes and susceptibility to xenobiotic stressors in the developing human liver.
The liver is the predominant organ of metabolism for many endogenous and foreign chemicals. Cytosolic sulfotransferases (SULTs) catalyze the sulfonation of drugs and other xenobiotics, as well as hormones, neurotransmitters, and sterols, with consequences that include enhanced drug elimination, hormone inactivation, and procarcinogen bioactivation. SULTs are classified into six gene families, but only SULT1 and SULT2 enzymes are expressed in human liver. We characterized the developmental expression patterns of SULT1 and SULT2 mRNAs and proteins in human liver samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing, and targeted quantitative proteomics. Using a set of prenatal, infant, and adult liver specimens, RT-qPCR analysis demonstrated that SULT1A1 (transcript variant 1) expression did not vary appreciably during development; SULT1C2, 1C4, and 1E1 mRNA levels were highest in prenatal and/or infant liver, and 1A2, 1B1, and 2A1 mRNA levels were highest in infant and/or adult. Hepatic SULT1A1 (transcript variant 5), 1C3, and 2B1 mRNA levels were low regardless of developmental stage. Results obtained with RNA sequencing of a different set of liver specimens (prenatal and pediatric) were generally comparable results to those of the RT-qPCR analysis, with the additional finding that SULT1A3 expression was highest during gestation. Analysis of SULT protein content in a library of human liver cytosols demonstrated that protein levels generally corresponded to the mRNAs, with the major exception that SULT1C4 protein levels were much lower than expected based on mRNA levels. These findings further support the concept that hepatic SULTs play important metabolic roles throughout the human life course, including early development.
Cytosolic sulfotransferase 1C3 (SULT1C3) is the least characterized of the three human SULT1C subfamily members. Originally identified as an orphan SULT by computational analysis of the human genome, we recently reported that SULT1C3 is expressed in human intestine and LS180 colorectal adenocarcinoma cells and is upregulated by agonists of peroxisome proliferator-activated receptor (PPAR) α and γ To determine the mechanism responsible for PPAR-mediated upregulation, we prepared reporter plasmids containing fragments of the SULT1C3 5'-flanking region. During initial attempts to amplify a 2.8-kb fragment from different sources of human genomic DNA, a 1.9-kb fragment was sometimes coamplified with the expected 2.8-kb fragment. Comparison of the 1.9-kb fragment sequence to the published SULT1C3 5'-flanking sequence revealed an 863-nt deletion (nt -146 to -1008 relative to the transcription start site). Transfection analysis in LS180 cells demonstrated that PPARα, δ, and γ agonist treatments induced luciferase expression from a reporter plasmid containing the 2.8-kb but not the 1.9-kb fragment. The PPAR agonists also activated a 1-kb reporter containing the 863-nt deletion region. Computational analysis identified three peroxisome proliferator response elements (PPREs) within the 863-nt region and serial deletions and site-directed mutations indicated that the most distal PPRE (at nt -769) was essential for obtaining PPAR-mediated transcriptional activation. Although agonists of all three PPARs could activate SULT1C3 transcription, RNA interference analysis indicated the predominance of PPARγ These data demonstrate that the PPARγ regulatory network includes SULT1C3 and imply that this enzyme contributes to the control of such PPARγ-regulated intestinal processes as growth, differentiation, and metabolism.
N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo-and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (V max ) of 11.1 nmol/min per mg and substrate affinity (K m ) of 41.6 mM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (V max /K m ) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC 50 values <50 mM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (k inact /K I ) of 0.68 and 0.51 minute, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or timedependent inhibition of CYP1A2 in the future clinical development of UTL-5g.
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