Tatsuki Fukami

Quantitative Analysis of UDP-Glucuronosyltransferase (UGT) 1A and UGT2B Expression Levels in Human Livers

UDP-glucuronosyltransferases (UGTs) catalyze glucuronidation of a variety of xenobiotics and endobiotics. UGTs are divided into two families, UGT1 and UGT2. The purpose of this study was to estimate the absolute expression levels of each UGT isoform in human liver and to evaluate the interindividual variability. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to determine the copy numbers of nine functional UGT1A isoforms and seven UGT2B isoforms. We noticed that not only primers but also templates as a standard for quantification should prudently be selected. Once we established appropriate conditions, the mRNA levels of each UGT isoform in 25 individual human livers were determined. UGT1A1 (0.9–138.5), UGT1A3 (0.1–66.6), UGT1A4 (0.1–143.3), UGT1A6 (1.0–70.4), UGT1A9 (0.3–132.4), UGT2B4 (0.3–615.0), UGT2B7 (0.2–97.4), UGT2B10 (0.7–253.2), UGT2B15 (0.3–107.8), and UGT2B17 (0.5–157.1) were substantially expressed (×104 copy/μg RNA) with large interindividual variability. Abundant isoforms were UGT2B4 and UGT2B10, followed by UGT1A1, UGT2B15, and UGT1A6. The sum of the UGT2B mRNA levels was higher than that of UGT1A mRNA levels. It is interesting to note that the mRNA levels normalized with glyceraldehyde-3-phosphate dehydrogenase mRNA for almost UGT isoforms that are substantially expressed in liver showed significant correlations to each other. Western blot analysis was performed using antibodies specific for UGT1A1, UGT1A4, UGT1A6, or UGT2B7. Correlation between the protein and mRNA levels was observed in only UGT1A1 (r = 0.488; p < 0.01). In conclusion, this study comprehensively determined the absolute values of mRNA expression of each UGT isoform in human livers and found considerable interindividual variability.

MicroRNAs Regulate Human Hepatocyte Nuclear Factor 4α, Modulating the Expression of Metabolic Enzymes and Cell Cycle

Hepatocyte nuclear factor (HNF) 4α is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4α. Potential recognition elements for miR-24 (MRE24) were identified in the coding region and the 3′-untranslated region (3′-UTR), and those for miR-34a (MRE34a) were identified in the 3′-UTR in HNF4α mRNA. The HNF4α protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4α mRNA level was significantly decreased by the overexpression of miR-24 but not by miR-34a. In luciferase analyses in HEK293 cells, the reporter activity of plasmid containing the 3′-UTR of HNF4α was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4α coding region downstream of the luciferase gene was significantly decreased by miR-24. These results suggest that the MRE24 in the coding region and MRE34a in the 3′-UTR are functional in the negative regulation by mRNA degradation and translational repression, respectively. The down-regulation of HNF4α by these microRNAs resulted in the decrease of various target genes such as cytochrome P450 7A1 and 8B1 as well as morphological changes and the decrease of the S phase population in HepG2 cells. We also clarified that the expressions of miR-24 and miR-34a were regulated by protein kinase C/mitogen-activated protein kinase and reactive oxygen species pathways, respectively. In conclusion, we found that human HNF4α was down-regulated by miR-24 and miR-34a, the expression of which are regulated by cellular stress, affecting the metabolism and cellular biology.

Human CYP2A6 is induced by estrogen via estrogen receptor.

Human CYP2A6, which is predominantly expressed in liver, is a key enzyme responsible for the metabolism of nicotine, coumarin, and some pharmaceutical drugs. CYP2A6 is also expressed in sex steroid-responsive tissues such as breast, ovary, uterus, testis, and adrenal grand. In this study, we examined the regulation of CYP2A6 gene by estrogen. Reverse transcription-polymerase chain reaction (RT-PCR) assays revealed that CYP2A6 mRNA was induced by estradiol in estrogen receptor (ER)-positive MCF-7 (2.9-fold) and HepG2 (1.3-fold) cells, but not in ER-negative MDA-MB-435 cells. Real-time RT-PCR assays revealed the CYP2A6 induction by estradiol in human hepatocytes (1.2- to 1.5-fold). Computer-assisted homology search identified a putative estrogen response element (ERE) at -2436 on the CYP2A6 gene. Electrophoretic mobility shift assays demonstrated specific binding of ERα to this element. Luciferase assays using MCF-7 cells revealed that the transcriptional activity of the CYP2A6 promoter was significantly activated by estradiol in an ERα-dependent manner, in which ERE was responsible for the activation. Chromatin immunoprecipitation assays verified the in vivo association of ERα with the ERE on the CYP2A6 gene. Immunohistochemical analyses using human endometrial tissues indicated that the CYP2A6 protein level in glandular cells was significantly higher in the proliferative phase than in the secretory phase, concomitant with local estrogen secretion during the menstrual cycle. These findings clearly demonstrated that CYP2A6 is directly induced by estrogen in an ERα-dependent manner, implying a biological role of CYP2A6 in estrogen-responsive tissues. Furthermore, this mechanism can also explain clinical aspects of increased nicotine metabolism under estrogen-rich environments.

PPARα Is Regulated by miR-21 and miR-27b in Human Liver

ABSTRACTPurposePeroxisome proliferator-activated receptor α (PPARα) is an important transcriptional factor that regulates genes encoding endo/xenobiotic enzymes and lipid metabolizing enzymes. In this study, we investigated whether microRNAs (miRNAs) are involved in the regulation of PPARα in human liver.MethodsPrecursor or antisense oligonucleotide for miR-21 or miR-27b was transfected into HuH7 cells; expression of PPARα and acyl-CoA synthetase M2B was determined by Western blot and real-time RT-PCR. Luciferase assay was performed to identify the functional miRNA recognition element (MRE). Expression levels of PPARα, miR-21, and miR-27b in a panel of 24 human livers were determined.ResultsThe overexpression and inhibition of miR-21 or miR-27b in HuH7 cells significantly decreased and increased the PPARα protein level, respectively, but not PPARα mRNA level. The miRNA-dependent regulation of PPARα affected the expression of its downstream gene. Luciferase assay identified a functional MRE for miR-21 in the 3′-untranslated region of PPARα. In human livers, the PPARα protein levels were not correlated with PPARα mRNA, but inversely correlated with the miR-21 levels, suggesting a substantial impact of miR-21, although the contribution of miR-27b could not be ruled out.ConclusionsWe found that PPARα in human liver is regulated by miRNAs.

A comprehensive review of UDP-glucuronosyltransferase and esterases for drug development

UDP-glucuronosyltransferase (UGT) and esterases are recognized as the most important non-P450 enzymes because of their high contribution to drug metabolism. UGTs catalyze the transfer of glucuronic acid to hydroxyl, carboxyl, or amine groups of compounds, whereas esterases hydrolyze compounds that contain ester, amide, and thioester bonds. These enzymes, in most cases, convert hydrophobic compounds to water-soluble metabolites to facilitate the elimination of compounds from the body. Information about these enzymes is steadily increasing, although our knowledge is still behind our understanding of P450. This review gives an overview of recent findings in UGT and esterases studies focusing on tissue distribution, gene regulation, substrate and inhibitor specificity, and species differences. In particular, the absolute protein content of UGT isoforms and esterases in human tissues could be available. In the field of esterases, it is becoming clear that enzymes other than carboxylesterase are involved in drug hydrolysis. In addition, there is an interesting interplay between UGTs and esterases in the formation and hydrolytic deglucuronidation of acyl-glucuronide, which is considered to be a reactive metabolite. With the growing awareness of the importance of non-P450 enzymes in drug development, issues that should be resolved are discussed.

CYP2A6 AND CYP2B6 ARE INVOLVED IN NORNICOTINE FORMATION FROM NICOTINE IN HUMANS: INTERINDIVIDUAL DIFFERENCES IN THESE CONTRIBUTIONS

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent Km = 173 ± 70 μM, Vmax = 57 ± 17 pmol/min/mg) and low-affinity (apparent Km = 619 ± 68 μM, Vmax = 137 ± 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent Km values of CYP2A6 (49 ± 12 μM) and CYP2B6 (550 ± 46 μM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 μM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 μM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.

Structure and characterization of human carboxylesterase 1a1 , 1a2 , and 1a3 genes

Objective Human carboxylesterase (CES) 1A1 gene (14 exons) and CES1A3 pseudogene (six exons) are inverted and duplicated genes in a reference sequence (NT_010498). In contrast, earlier studies reported the CES1A2 gene (14 exons) instead of the CES1A3 pseudogene. The sequences of the CES1A2 gene downstream and upstream of intron 1 are identical with those of the CES1A1 and CES1A3 genes, respectively. A CES1A1 variant of which exon 1 is converted with that of the CES1A3 gene (the transcript is CES1A2) has recently been identified. We sought to clarify the confusing gene structure of human CES1A. Methods A panel of 55 human liver as well as 318 blood samples (104 Caucasians, 107 African–Americans, and 107 Japanese) was used to clarify the gene structures of CES1A1, CES1A2, and CES1A3. Real-time reverse transcription-PCR and western blot analysis were carried out. Imidapril hydrolase activity in human liver microsomes and cytosol was determined by liquid chromatography-mass spectrometry (LC-MS)/MS. Results By PCR analyses, we found that the CES1A2 gene is a variant of the CES1A3 gene. Four haplotypes, A (CES1A1 wild type and CES1A3), B (CES1A1 wild type and CES1A2), C (CES1A1 variant and CES1A3), and D (CES1A1 variant and CES1A2), were demonstrated. Ethnic differences were observed in allele frequencies of CES1A1 variant (17.3% in Caucasians and African–Americans and 25.2% in Japanese) and CES1A2 gene (14.4% in Caucasians, 5.1% in African–Americans, and 31.3% in Japanese). In human livers whose diplotype was A/A and C/C or C/D, no CES1A2 and CES1A1 mRNA was detected, respectively. In the other participants, the CES1A1 mRNA levels were higher than the CES1A2 mRNA levels. The CES1A proteins translated from CES1A1 mRNA and CES1A2 mRNA were detected in both human liver microsomes and cytosol fractions suggesting that the differences in exon 1 encoding a signal peptide did not affect the subcellular localization. Imidapril hydrolase activities reflected the CES1A protein levels. Conclusion We found that the CES1A2 gene is a variant of the CES1A3 pseudogene. The findings presented here significantly increase our understanding about the gene structure and expression properties of human CES1A.

CYP2A13 expressed in human bladder metabolically activates 4-aminobiphenyl

Cigarette smoking is the predominant risk factor for bladder cancer. Aromatic amines such as 4‐aminobiphenyl (ABP) is the major carcinogens found in tobacco smoke. Although it is generally accepted that ABP is metabolically activated via N‐hydroxylation by CYP1A2 in human liver, previous studies using Cyp1a2‐null mice indicated the involvement of other enzyme(s). Here we found that CYP2A13 can metabolically activate ABP to show genotoxicity by Umu assay. The Km and Vmax values for ABP N‐hydroxylation by recombinant CYP2A13 in E. coli were 38.5 ± 0.6 μM and 7.8 ± 0.0 pmol/min/pmol CYP, respectively. The Km and Vmax values by recombinant CYP1A2 were 9.9 ± 0.9 μM and 39.6 ± 0.9 pmol/min/pmol CYP, respectively, showing 20‐fold higher intrinsic clearance than CYP2A13. In human bladder, CYP2A13 mRNA, but not CYP1A2, is expressed at a relatively high level. Human bladder microsomes showed ABP N‐hydroxylase activity (Km = 34.9 ± 4.7 μM and Vmax = 57.5 ± 1.9 pmol/min/mg protein), although the intrinsic clearance was 5‐fold lower than that in human liver microsomes (Km = 33.2 ± 2.0 μM and Vmax = 293.9 ± 5.8 pmol/min/mg protein). The activity in human bladder microsomes was prominently inhibited by 8‐methoxypsoralen, but not by fluvoxamine, anti‐CYP1A2 or anti‐CYP2A6 antibodies. CYP2S1, which is expressed in human bladder and has relatively high amino acid identities with CYP2As, did not show detectable ABP N‐hydroxylase activity. In conclusion, although the enzyme responsible for ABP N‐hydroxylation in human bladder microsomes could not be determined, we found that CYP2A13 metabolically activates ABP.

Human arylacetamide deacetylase is responsible for deacetylation of rifamycins: rifampicin, rifabutin, and rifapentine.

Rifamycins such as rifampicin, rifabutin, and rifapentine are used for the treatment of tuberculosis and induce various drug-metabolizing enzymes. Rifamycins have been reported to be mainly deacetylated by esterase(s) expressed in human liver microsomes (HLM) to 25-deacetylrifamycins, but the responsible enzyme remained to be determined. In this study, we found that recombinant human arylacetamide deacetylase (AADAC) could efficiently deacetylate rifamycins, whereas human carboxylesterases, which are enzymes responsible for the hydrolysis of many prodrugs, showed no activity. The involvement of AADAC in the deacetylation of rifamycins in HLM was verified by the similarities of the K(m) and K(i) values and the inhibitory characteristics between recombinant AADAC and HLM. Rifamycins exhibited potent cytotoxicity to HepG2 cells, but their 25-deacetylated metabolites did not. Luciferase assay using a reporter plasmid containing CYP3A4 direct repeat 3 and everted repeat 6 motifs revealed that 25-deacetylrifamycins have lesser potency to transactivate CYP3A4 compared with the parent drugs. Supporting these results, HepG2 cells infected with a recombinant adenovirus expressing human AADAC showed low cytotoxicity and induction potency of CYP3A4 by rifamycins. In addition, CYP3A4 induction in human hepatocytes by rifamycins was increased by transfecting siRNA for human AADAC. Thus, we found that human AADAC was the enzyme responsible for the deacetylation of rifamycins and would affect the induction rate of drug-metabolizing enzymes by rifamycins and their induced hepatotoxicity.

Plasma MicroRNA Profiles in Rat Models of Hepatocellular Injury, Cholestasis, and Steatosis

MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121–317 miRNAs) were increased over 2-fold and the levels of a small number of miRNAs (6–35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.