Masataka Takamiya

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.

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.

Human arylacetamide deacetylase is a principal enzyme in flutamide hydrolysis.

Flutamide, an antiandrogen drug, is widely used for the treatment of prostate cancer. The initial metabolic pathways of flutamide are hydroxylation and hydrolysis. It was recently reported that the hydrolyzed product, 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1), is further metabolized to N-hydroxy FLU-1, an assumed hepatotoxicant. However, the esterase responsible for the flutamide hydrolysis has not been characterized. In the present study, we found that human arylacetamide deacetylase (AADAC) efficiently hydrolyzed flutamide using recombinant AADAC expressed in COS7 cells. In contrast, carboxylesterase1 (CES1) and CES2, which are responsible for the hydrolysis of many drugs, could not hydrolyze flutamide. AADAC is specifically expressed in the endoplasmic reticulum. Flutamide hydrolase activity was highly detected in human liver microsomes (Km, 794 ± 83 μM; Vmax, 1.1 ± 0.0 nmol/min/mg protein), whereas the activity was extremely low in human liver cytosol. The flutamide hydrolase activity in human liver microsomes was strongly inhibited by bis-(nonylphenyl)-phenylphosphate, diisopropylphosphorofluoride, and physostigmine sulfate (eserine) but moderately inhibited by sodium fluoride, phenylmethylsulfonyl fluoride, and disulfiram. The same inhibition pattern was obtained with the recombinant AADAC. Moreover, human liver and jejunum microsomes showing AADAC expression could hydrolyze flutamide, but human pulmonary and renal microsomes, which do not express AADAC, showed slight activity. In human liver microsomal samples (n = 50), the flutamide hydrolase activities were significantly correlated with the expression levels of AADAC protein (r = 0.66, p < 0.001). In conclusion, these results clearly showed that flutamide is exclusively hydrolyzed by AADAC. AADAC would be an important enzyme responsible for flutamide-induced hepatotoxicity.

Aryl hydrocarbon receptor nuclear translocator in human liver is regulated by miR-24.

Aryl hydrocarbon receptor nuclear translocator (ARNT) forms a heterodimer with aryl hydrocarbon receptor or hypoxia inducible factor 1α to mediate biological responses to xenobiotic exposure and hypoxia. Although the regulation mechanism of the ARNT expression is largely unknown, earlier studies reported that the human ARNT protein level was decreased by hydrogen peroxide or reactive oxygen species. These stimuli increase the miR-24 level in various human cell lines. In silico analysis predicts that some microRNAs including miR-16 and miR-23b may bind to ARNT mRNA. This background prompted us to investigate whether human ARNT is regulated by microRNAs. Overexpression of miR-24 into HuH-7 and HepG2 cells significantly decreased the ARNT protein level, but not the ARNT mRNA level, indicating translational repression. However, overexpression of miR-16 or miR-23b caused no change in the ARNT expression. The miR-24-dependent down-regulation of ARNT decreased the expression of its downstream genes such as CYP1A1 and carbonic anhydrase IX. Luciferase assay was performed to determine the element on the ARNT mRNA to which miR-24 binds. Finally, it was demonstrated that the miR-24 levels in a panel of 26 human livers were inversely correlated with the protein levels or the translational efficiency of ARNT. Taken together, we found that miR-24 negatively regulates ARNT expression in human liver, affecting the expression of its downstream genes. miR-24 would be one of the factors underlying the mechanisms by which ARNT protein is decreased by reactive oxygen species.

A histological study on the mechanism of epidermal nuclear elongation in electrical and burn injuries.

Abstract Epidermal nuclear elongation is one of the most important signs for the diagnosis of electrical injury. In this study, we investigated the mechanism responsible for this phenomenon by comparing the findings from burn injuries and those from contusions. Electrical and burn injuries were made in the dorsal skin of rats using energy ranging from 100 to 790 joules for electrical injury, and 170–690 joules for burn injury. Contusions were also made by compressing the skin with a vice. In electrical and burn injuries, the dermis under the epidermal elongated nuclei was homogeneous and without empty spaces between collagen bundles and the number of dermal fibroblasts per 0.01 mm2 below the damaged epidermis decreased significantly (P < 0.05). The incidence of this change correlated with the depth of denatured dermal collagen fibres and in both types of injuries, dermal cells had no nuclear antigenicity for ubiquitin. The width of the injured epidermis with nuclear elongation decreased significantly (P < 0.05) and the elongated nuclei were parallel to the basal membrane. In electrical injury however, nuclear elongation occurred more frequently near the external root sheath. Nuclear elongation of fibroblasts and external root sheath cells was also found, but those of sebaceous gland cells were not detected. Epidermal elongated nuclei were also found in contusions. The evidence strongly suggests that epidermal nuclear elongation in electrical and burn injuries is due to dermal expansion by heat.

Wound age estimation by simultaneous detection of 9 cytokines in human dermal wounds with a multiplex bead-based immunoassay: An estimative method using outsourced examinations

Wound age estimation for human dermal wounds was performed based on quantification of interleukin 1beta (IL 1beta), IL 5, IL 7, IL 12 p70, IL 13, IL 17, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP 1), and macrophage inflammatory protein 1beta (MIP 1beta). IL 5, IL 12 p 70, IL 13, and IL 17 increased from the early phase, MCP 1 exclusively in the middle phase, and IL 1beta, G-CSF, and MIP 1beta from the middle phase to the late phase. IL 7 decreased from the early phase. Among the cytokines analyzed in the present study, MCP 1 was the most plentiful cytokine. In addition, an outsourced examination, which could be available to any forensic institute, was performed in two cases for confirmative purposes. Many factors have been proposed as markers for dermal wound age estimation, but the set of cytokines selected for the outsourced examination in the present study wound be useful in daily forensic practice.

A case of acute gasoline intoxication at the scene of washing a petrol tank.

We encountered a case of acute gasoline intoxication at the scene of washing the inner wall of a petrol tank. The decedent was a 50-year-old male, who was the supervisor. Two young workers wearing mask respirators began to wash the inner wall of the gasoline tank under poor ventilation. About 1 h later, because one of the workers lost consciousness, the supervisor entered the tank, without a mask respirator, to rescue the worker. However, the supervisor immediately fainted, and died 26 h after the accident. In the autopsy, blisters were observed on the skin of the face, neck, anterior chest, upper extremities, and back. The heart contained hemolyzed blood. Histologically, hemorrhagic pulmonary edema, pneumonia, and proximal tubular necrosis were found. In the toxicological analyzes, toluene, xylene, and trimethylbenzene were detected in the blood, brain, and gastric contents. Toluene concentrations in the blood and brain were 0.3 mug/ml and 3.7 mug/g, respectively. Since pathological findings were consistent with the reported findings concerning gasoline intoxication, and constituents of gasoline were in the body, death was attributed to acute gasoline intoxication. It was considered that sufficient ventilation and proper use of a mask respirator were essential for this kind of work.

CYP2A7 Pseudogene Transcript Affects CYP2A6 Expression in Human Liver by Acting as a Decoy for miR-126*

Human cytochrome P450 (CYP)2A6 is responsible for the metabolic activation of tobacco-related nitrosamines, as well as the metabolism of nicotine and some pharmaceutical drugs. There are large interindividual differences in CYP2A6 activity and expression, largely attributed to genetic polymorphisms. However, the variability was observed within homozygotes of the wild-type CYP2A6 gene. In this study, we investigated the possibility that CYP2A6 might be regulated by microRNA. A luciferase assay revealed that a microRNA recognition element (MRE) of miR-126* found in the 3′-untranslated region (UTR) of CYP2A6 mRNA is functional. We established two HEK293 cell lines stably expressing CYP2A6, with one including and the other excluding the full-length 3′-UTR (HEK/2A6+UTR and HEK/2A6 cells, respectively). Overexpression of miR-126* markedly decreased CYP2A6 protein levels, enzyme activity, and mRNA level in HEK/2A6+UTR cells, whereas it marginally decreased those in HEK/2A6 cells, indicating that the 3′-UTR including the MRE is functional for the downregulation of CYP2A6 by miR-126*. The inhibition of miR-126* increased CYP2A6 protein levels in primary human hepatocytes, suggesting that miR-126* downregulates endogenous CYP2A6 expression. In 20 human liver samples, the expression ratios of CYP2A6 and a pseudogene transcript CYP2A7 mRNA were highly variable (CYP2A7/CYP2A6: 0.1 to 12). Interestingly, we found that CYP2A7 was another target of miR-126* and restored the miR-126*–dependent downregulation of CYP2A6 by acting as a decoy for miR-126*. In conclusion, this study demonstrates that human CYP2A6 is post-transcriptionally regulated by miR-126* and that CYP2A7 affects CYP2A6 expression by competing for miR-126* binding.

Single-Nucleotide Polymorphisms in Cytochrome P450 2E1 (CYP2E1) 3′-Untranslated Region Affect the Regulation of CYP2E1 by miR-570

Human cytochrome P450 2E1 (CYP2E1) catalyzes the metabolism of numerous xenobiotics, including acetaminophen and ethanol. CYP2E1 expression is known to be extensively regulated by post-transcriptional and post-translational mechanisms. A previous study had reported that a single-nucleotide polymorphism (SNP) 1561A>G in the 3′-untranslated region (3′-UTR) of CYP2E1 leads to a decreased CYP2E1 mRNA level in human peripheral blood mononuclear cells. In this study, we examined the possibility that microRNA(s) (miR) may be involved in the SNP-mediated modulation of CYP2E1 expression. Genotyping and sequencing analyses revealed that another SNP, 1556T>A, in the 3′-UTR was in complete linkage disequilibrium with the SNP 1561A>G. We termed the alleles with 1556T and 1561A or 1556A and 1561G haplotype I or II, respectively. A luciferase assay revealed that miR-570 recognizes the CYP2E1 3′-UTR of haplotype I but not haplotype II. Human embryonic kidney 293 (HEK293) cell lines stably expressing human CYP2E1 that included the 3′-UTR of haplotype I or II (HEK/2E1(I) or HEK/2E1(II) cells, respectively) were established. Overexpression of miR-570 significantly decreased the CYP2E1 protein level in the HEK/2E1(I) cells but not in the HEK/2E1(II) cells. In seven human livers with diplotype I/I, the CYP2E1 protein levels were inversely correlated with the miR-570 levels, but no relationship was observed in 25 human livers with diplotypes I/II and II/II. Collectively, it was demonstrated that human CYP2E1 was regulated by miR-570 in a genotype-dependent manner. This report describes the first proof that SNP in 3′-UTR of human P450 affects binding of miRNA to modulate the expression in the liver.