Bile acids are synthesized from cholesterol in the liver and subjected to multiple metabolic biotransformations in hepatocytes, including oxidation by cytochromes P450 (CYPs) and conjugation with taurine, glycine, glucuronic acid, and sulfate. Mice and rats can hydroxylate chenodeoxycholic acid (CDCA) at the 6β-position to form α-muricholic acid (MCA) and ursodeoxycholic acid (UDCA) to form β-MCA. However, MCA is not formed in humans to any appreciable degree and the mechanism for this species difference is not known. Comparison of several Cyp-null mouse lines revealed that α-MCA and β-MCA were not detected in the liver samples from Cyp2c-cluster null (Cyp2c-null) mice. Global bile acid analysis further revealed the absence of MCAs and their conjugated derivatives, and high concentrations of CDCA and UDCA in Cyp2c-null mouse cecum and feces. Analysis of recombinant CYPs revealed that α-MCA and β-MCA were produced by oxidation of CDCA and UDCA by Cyp2c70, respectively. CYP2C9-humanized mice have similar bile acid metabolites as the Cyp2c-null mice, indicating that human CYP2C9 does not oxidize CDCA and UDCA, thus explaining the species differences in production of MCA. Because humans do not produce MCA, they lack tauro-β-MCA, a farnesoid X receptor antagonist in mouse that modulates obesity, insulin resistance, and hepatosteatosis.

In this review, novel aspects of the role of esterases, which contribute to the metabolism of 10% of therapeutic drugs, are described. Esterases hydrolyze the compounds that contain ester, amide, and thioester bonds, which cause prodrug activation or detoxification. Among esterases, carboxylesterases are well known to be involved in the hydrolysis of a variety of drugs. Additionally, other esterases have recently received attention for their pharmacological and toxicological roles. Arylacetamide deacetylase (AADAC) is involved in the hydrolysis of flutamide, phenacetin, and rifamycins. AADAC is associated with adverse drug reactions because the hydrolytic metabolites of flutamide and phenacetin appear to be associated with hepatotoxicity and nephrotoxicity/hematotoxicity, respectively. Paraoxonase and butyrylcholinesterase hydrolyze pirocarpine/simvastatin and succinylcholine/bambuterol, respectively. Although the esterases that hydrolyze the acyl-glucuronides of drugs have largely been unknown, we recently found that α/β hydrolase domain containing 10 (ABHD10) is responsible for the hydrolysis of mycophenolic acid acyl-glucuronide in human liver. Because acyl-glucuronides are associated with toxicity, ABHD10 might function as a detoxification enzyme. Thus, various esterases, which include enzymes that have not been known to hydrolyze drugs, are involved in drug metabolism with different substrate specificity. Further esterase studies should be conducted to promote our understanding in clinical pharmacotherapy and drug development.

Human cytochrome P450 (CYP) 2A6 metabolizes nicotine to cotinine and is a possible modulator of nicotine addiction. Quantitative and qualitative differences in nicotine addiction have been observed between ethnic groups. However, there are few data on the ethnic influences of the CYP2A6-nicotine metabolism relationship, particularly with regard to black subjects. We determined the nicotine metabolism and CYP2A6 genotype in 176 white subjects and 160 black subjects, comparing them with our previous data from 209 Korean subjects and 92 Japanese subjects. Large interindividual differences were observed in the cotinine/nicotine ratios in plasma calculated as an index of nicotine metabolism in white subjects (range, 0.6-36.5) and in black subjects (range, 0.9-30.4). No ethnic difference in the metabolic ratio was observed among white subjects (mean, 7.2 +/- 5.0), black subjects (mean, 7.1 +/- 4.7), and Korean subjects (mean, 8.7 +/- 11.9), whereas Japanese subjects showed a significantly (P < .005) lower metabolic ratio (mean, 3.8 +/- 3.1) compared with the other populations. Women showed significantly (P < .05) higher metabolic ratios than men in the black population (8.0 +/- 5.3 versus 6.0 +/- 3.7). Obvious ethnic differences in the CYP2A6 alleles were observed among these 4 populations. The combined frequencies of the alleles lacking or showing reduced enzymatic activity (CYP2A6*2, CYP2A6*4, CYP2A6*5, CYP2A6*7, CYP2A6*9, CYP2A6*10, CYP2A6*11, CYP2A6*17, CYP2A6*19, and CYP2A6*20) were 9.1%, 21.9%, 42.9%, and 50.5% in white, black, Korean, and Japanese subjects, respectively. These CYP2A6 alleles were associated with reduced nicotine metabolism. Among the homozygotes of CYP2A6*1, interindividual and ethnic differences in the metabolic ratio were still observed. Thus some factors other than genetic ones might also contribute to the interindividual and ethnic differences. This comprehensive study of 4 populations extends our understanding of nicotine metabolism and the impact of genetic polymorphisms of the CYP2A6 gene.

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.

Human CYP2E1 is one of the pharmacologically and toxicologically important cytochrome P450 isoforms. Earlier studies have reported that the CYP2E1 expression is extensively regulated by post-transcriptional and post-translational mechanisms, but the molecular basis remains unclear. In the present study, we examined the possibility that microRNA may be involved in the post-transcriptional regulation of human CYP2E1. In silico analysis identified a potential recognition element of miR-378 (MRE378) in the 3'-untranslated region (UTR) of human CYP2E1 mRNA. Luciferase assays using HEK293 cells revealed that the reporter activity of the plasmid containing the MRE378 was decreased by co-transfection of precursor miR-378, indicating that miR-378 functionally recognized the MRE378. We established two HEK293 cell lines stably expressing human CYP2E1 including or excluding 3'-UTR. When the precursor miR-378 was transfected into the cells expressing human CYP2E1 including 3'-UTR, the CYP2E1 protein level and chlorzoxazone 6-hydroxylase activity were significantly decreased, but were not in the cells expressing CYP2E1 excluding 3'-UTR. In both cell lines, the CYP2E1 mRNA levels were decreased by overexpression of miR-378, but miR-378 did not affect the stability of CYP2E1 mRNA. In a panel of 25 human livers, no positive correlation was observed between the CYP2E1 protein and CYP2E1 mRNA levels, supporting the post-transcriptional regulation. Interestingly, the miR-378 levels were inversely correlated with the CYP2E1 protein levels and the translational efficiency of CYP2E1. In conclusion, we found that human CYP2E1 expression is regulated by miR-378, mainly via translational repression. This study could provide new insight into the unsolved mechanism of the post-transcriptional regulation of CYP2E1.