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Toxoplasma gondii is an intracellular protozoan parasite causing toxoplasmosis, an infectious disease affecting warm-blooded vertebrates worldwide. Many drug-metabolizing enzymes are located in the liver, a major organ of drug metabolism, and their function can be affected by pathogen infection. Using next-generation sequencing (RNA-seq) and quantitative polymerase chain reaction (qPCR), changes in the hepatic expressions of drug-metabolizing enzymes were analysed in mice chronically infected with T. gondii. The analysis found that, among drug-metabolizing enzymes, 22 genes were upregulated and 28 genes were downregulated (≥1.5-fold); of these 5 and 17 genes, respectively, were cytochromes P450 (Cyp or P450). Subsequent qPCR analysis showed that six P450 genes were upregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp1b1, Cyp2c29, Cyp2c65, Cyp2d9, Cyp2d12, and Cyp3a59, whereas nine P450 genes were downregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c69, Cyp2d40, Cyp2e1, Cyp3a11, Cyp3a41, and Cyp3a44. Moreover, metabolic assays in infected mouse liver using typical P450 substrates revealed that midazolam 1′-hydroxylation and testosterone 2-hydroxylation activities decreased significantly (≥1.5-fold, p < 0.05), whereas testosterone 16-hydroxylation activity increased significantly (≥1.5-fold, p < 0.05). Chronic Toxoplasma infection affects drug metabolism, at least partly, by altering the gene expressions of drug-metabolizing enzymes, including P450s. [ABSTRACT FROM AUTHOR]

Chimeric TK-NOG mice with a humanized liver (normal Hu-liver) are a unique animal model for predicting drug metabolism in humans. However, residual mouse hepatocytes occasionally prevent the precise evaluation of human drug metabolism. Herein, we developed a novel humanized liver TK-NOG mouse with a conditional knockout of liver-specific cytochrome P450 oxidoreductase (POR cKO Hu-liver). Immunohistochemical analysis revealed only a few POR-expressing cells around the portal vein in POR cKO mouse livers. NADPH-cytochrome c reductase and cytochrome P450 (P450)-mediated drug oxidation activity in liver microsomes from POR cKO mice was negligible. After the intravenous administration of S-warfarin, high circulating and urinary levels of S-7-hydroxywarfarin (a major human metabolite) were observed in POR cKO Hu-liver mice. Notably, the circulating and urinary levels of S-4′-hydroxywarfarin (a major warfarin metabolite in mice) were much lower in POR cKO Hu-liver mice than in normal Hu-liver mice. POR cKO Hu-liver mice with minimal interference from mouse hepatic P450 oxidation activity are a valuable model for predicting human drug metabolism. [ABSTRACT FROM AUTHOR]

Pigs are an important species used in drug metabolism studies; however, the cytochromes P450 (P450s or CYPs) have not been fully investigated in pigs. In this study, pig CYP2C32, CYP2C33, CYP2C34, CYP2C36, CYP2C42, and CYP2C49 cDNAs were isolated and found to contain open reading frames of 490 or 494 amino acids that shared 64–82% sequence identity with human CYP2C8/9/18/19. Pig CYP2C genes formed a gene cluster in a genomic region that corresponded to that of the human CYP2C cluster; an additional gene cluster was formed by pig CYP2C33a and CYP2C33b distant from the first cluster but located in the same chromosome. Among the tissues analysed, these pig CYP2C mRNAs were preferentially expressed in liver, small intestine, and/or kidney; pig CYP2C49, CYP2C32, CYP2C34, and CYP2C33 mRNAs were the most abundant CYP2C mRNAs in liver, jejunum, ileum, and kidney, respectively. Metabolic assays showed that pig CYP2C proteins (heterologously expressed in Escherichia coli) metabolised typical human CYP2C substrates diclofenac, warfarin, and/or omeprazole. The results suggest that these pig CYP2Cs are functional enzymes able to metabolise human CYP2C substrates in liver and small intestine, just as human CYP2Cs do. [ABSTRACT FROM AUTHOR]

Despite the importance of mice as a preclinical species in drug testing, their hepatic and extrahepatic drug-metabolising characteristics are poorly understood. Here, we compared the P450-dependent drug oxidation activity in tissue microsomes and distribution patterns of P450 protein/mRNA between humans and mice. The activities of midazolam 1′-/4-hydroxylation in the liver and intestine and chlorzoxazone 6-hydroxylation in the liver were similar in humans and mice. The activities of coumarin 7-hydroxylation, flurbiprofen 4′-hydroxylation, and S-mephenytoin 4′-hydroxylation in the liver were higher in humans than in mice. The activities of 7-ethoxyresorufin O-deethylation in the liver, 7-pentoxyresorufin O-depentylation in the lung/liver/intestine, bufuralol 1′-hydroxylation in the liver/intestine, propafenone 4′-hydroxylation in liver/intestine, and diazepam N-demethylation in the liver/intestine were higher in mice than in humans. CYP1A2/2E1 mRNAs were mainly expressed in the livers of humans and mice. Cyp2b9/2b10 mRNAs were abundant in the mouse lung/liver/intestine, but CYP2B6 was mainly expressed in the human liver. CYP2C/2D/3A mRNAs were expressed in the liver and intestine, with the respective proteins detected in tissue microsomes of both humans and mice. These information on P450-dependent drug-metabolising characteristics in hepatic and extrahepatic tissues is useful to understand the similarities and differences between humans and mice in drug metabolism. [ABSTRACT FROM AUTHOR]

The cynomolgus macaque is a non-human primate species widely used in drug metabolism studies. Despite the importance of genetic polymorphisms in cytosolic aldehyde oxidase (AOX) 1 in humans, genetic variants have not been investigated in cynomolgus or rhesus macaques. Genetic variants in AOX1 were identified and allele frequencies were assessed using the genomes of 24 cynomolgus and 8 rhesus macaques. The analysis identified 38 non-synonymous variants, some of which were unique to cynomolgus macaques (bred in Cambodia, Indochina, or Indonesia) or rhesus macaques, whereas many variants were shared by the two lineages. Among the variants observed at relatively high frequencies, eight were selected for functional analysis. Recombinant P605L and V1338I AOX1 variants showed substantially lower phthalazine and carbazeran oxidation activities than the wild-type AOX1 protein. In liver cytosolic fractions from cynomolgus and rhesus macaques genotyped for P605L and V1338I AOX1, groups of cytosolic fractions with P605L and/or V1338I AOX1 variants showed significantly lower phthalazine and carbazeran oxidation activities than the wild type. These results indicate that AOX1 is polymorphic in cynomolgus and rhesus macaques, just as it is in humans. Further investigation is needed to reveal the functional significance of these AOX1 variants in drug metabolism. [ABSTRACT FROM AUTHOR]

1. Although the cynomolgus macaque is an important non-human primate species used in drug metabolism studies, cynomolgus macaque microRNA expressions have not been fully investigated. 2. The expressions of 11 cynomolgus microRNAs, all orthologues of P450 regulators in humans, were measured by quantitative polymerase chain reaction in adrenal gland, brain, heart, jejunum, kidney, liver, ovary, testis and uterus. mfa-miR-122 and mfa-miR-192, potentially important biomarkers for liver toxicity, were also analyzed. 3. Several cynomolgus microRNAs showed preferential tissue expressions: mfa-miR-1 in heart, mfa-miR-122 in liver and mfa-miR-21 and mfa-miR-192 in jejunum. The remaining nine microRNAs had more ubiquitous expressions. All 13 cynomolgus microRNAs were expressed in liver. Among the 10 animals investigated, inter-individual microRNA expression levels in liver varied from 1.5- to 5.3-fold. mfa-miR-18b was the most variable microRNA. Sex differences in expression levels were <2.0-fold, and the difference was only significant for mfa-miR-29 [1.6-fold difference (p <.05)]. Six cynomolgus microRNAs (mfa-miR-18b, mfa-miR-27a, mfa-miR-132, mfa-miR-27b, mfa-miR-122 and mfa-miR-29) were significantly correlated with P450 mRNAs: mfa-miR-18b and mfa-miR-27a were each correlated with seven P450 mRNAs. 4. Expression of these cynomolgus microRNAs in liver might indicate their possible roles in this tissue, and further investigation will clarify their involvement in P450 regulation. [ABSTRACT FROM AUTHOR]

Glutathione S-transferase (GST) is a family of enzymes important for conjugation with glutathione of endogenous and exogenous compounds. Human GSTM1 null allele is associated with toxicity and cancers. Cynomolgus and rhesus macaques have molecular and enzymatic similarities of GSTs to humans; however, genetic variants have not been investigated. In macaques, instead of pseudogenized GSTM1, GSTM5 is a predominant GSTM isoform. In this study, re-sequencing of GSTM5 in 64 cynomolgus and 31 rhesus macaques found 6 non-synonymous variants, and 1 variant (IVS5 + 1) causing exon skip. Of these 7 variants, 3 and 1 were found only in Indochinese and Indonesian cynomolgus macaques, respectively. Cynomolgus GSTM5-mediated styrene 7,8-oxide and trans-stilbene oxide conjugation activities correlated with GSTM protein levels immunochemically quantified in cynomolgus liver samples. Using recombinant GSTM5 proteins, 4 of the 6 non-synonymous variants including E29Q, L96R, M166V and S201N showed substantially lower metabolic activities. Moreover, a homozygote for E29Q and heterozygotes for S201N or IVS5 + 1 showed significantly lower conjugation activities in liver cytosolic fractions as compared with wild-type animals. Therefore, the present results suggest that inter-animal variability of GST-dependent drug metabolism is at least partly accounted for by GSTM5 variants in cynomolgus and rhesus macaques as pre-clinical animal models. [ABSTRACT FROM AUTHOR]

The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low Km value was relatively comparable to that for marmoset livers. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers. [ABSTRACT FROM AUTHOR]

1. Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined. 2. The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6β-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1′-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2 C and 3 A antibodies suppressed progesterone 21/17α- and 16α/6β-hydroxylation, respectively, in marmoset liver microsomes. 3. Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/Km values of 0.53 and 0.089 mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6β-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients). 4. These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6β-hydroxylation by marmoset P450 2 C and 3 A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets. [ABSTRACT FROM AUTHOR]

1. Cytochrome P450 (P450) regulators including nuclear receptors and transcription factors have not been fully investigated in cynomolgus macaques, an important species used in drug metabolism studies. In this study, we analyzed 17 P450 regulators by sequence and phylogenetic analysis, and tissue expression. 2. Gene and genome structures of 17 P450 regulators were similar to the human orthologs, and the deduced amino acid sequences showed high sequence identities (92-95%) and more closely clustered in a phylogenetic tree, with the human orthologs. 3. Many of the P450 regulator mRNAs were preferentially expressed in the liver, kidney, and/or jejunum. Among the P450 regulator mRNAs, PXR was most abundant in the liver and jejunum, and HNF4α in the kidney. In the liver, the expression of most P450 regulator mRNAs did not show significant differential expression (>2.5-fold) between cynomolgus macaques bred in Cambodia, China, and Indonesia, or rhesus macaques. 4. By correlation analysis, most of the P450 regulators were significantly (p < 0.05) correlated to other P450 regulators, and many of them were also significantly (p < 0.05) correlated with P450s. 5. These results suggest that 17 P450 regulators of cynomolgus macaques had similar molecular characteristics to the human orthologs. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (P450) largely remain to be characterized in great apes. Comparative immunochemical detection of drug metabolizing forms of P450s 1A, 2A, 2B, 2C, 2D, 2E, 2J, 3A, 4A, and 4F in liver microsomes from chimpanzees, gorillas, orangutans, gibbons, cynomolgus and rhesus macaques, and common marmosets were carried out. [ABSTRACT FROM AUTHOR]

1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity – attributable to metabolites formed by cytochrome P450 enzymes – but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylationin vivo.Introducing one aromatic amino group into thalidomide resulted in less species differences inin vivopharmacokinetics in control and humanized-liver mice. [ABSTRACT FROM AUTHOR]

1. Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes. 2. Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice. 3. Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver. 4. Enzyme assays using marmoset P450 2J2 protein heterologously expressed inEscherichia coliindicated that marmoset P450 2J2 effectively catalyzed astemizoleO-demethylation and terfenadinet-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes. 5. These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans. [ABSTRACT FROM PUBLISHER]

The pharmacokinetics of cytochrome P450 probes in humans can be extrapolated from corresponding data in cynomolgus monkeys using simplified physiologically based pharmacokinetic (PBPK) modeling. In the current study, despite some species difference in drug clearances, this modeling methodology was adapted to estimate human plasma concentrations of P450 probes based on data from commonly used medium-sized experimental animals, namely dogs and minipigs.Using known species allometric scaling factors and in vitro metabolic clearance data, the observed plasma concentrations of slowly eliminated caffeine and warfarin and rapidly eliminated omeprazole, metoprolol and midazolam in two young dogs were scaled to human oral monitoring equivalents. Using the same approach, the previously reported pharmacokinetics of the five P450 probes in minipigs was also scaled to human monitoring equivalents.The human plasma concentration profiles of the five P450 probes estimated by the simplified human PBPK models based on observed/reported pharmacokinetics in dogs/minipigs were consistent with previously published pharmacokinetic data in humans.These results suggest that dogs and minipigs, in addition to monkeys, could be suitable models for humans during research into new drugs, especially when used in combination with simple PBPK models. [ABSTRACT FROM AUTHOR]

Aim The hepatoma-derived cell line HepaRG is regarded as an in vitro model of drug metabolism because fully differentiated HepaRG cells demonstrate functional metabolic responses comparable to those of primary human hepatocytes. Recently, it was demonstrated that the 3D culture of HepaRG cells enhanced their metabolic functions and toxicological responses. We approached the mechanisms underlying these enhancement effects. Methods We compared 2D-cultured HepaRG cells with 3D-cultured HepaRG spheroids in the gene expression patterns and the metabolic functions. In the present study, we performed 3D culture of HepaRG cells using functional polymers (FP). To reveal the in vivo differentiation ability, we transplanted the 3D-cultured HepaRG spheroids into TK-NOG mice. Results A comparison between 2D and 3D cultures revealed that 3D-cultured HepaRG spheroids demonstrated reductions in bile duct marker expression, accelerated expression of cytochrome P450 3A4, and increases in the ratio of albumin-expressing hepatocytes. Furthermore, catalytic activities of cytochrome P450 3A4 were modified by omeprazole and rifampicin in the 3D-cultured HepaRG spheroids. Transplantation analysis revealed that 3D-cultured HepaRG spheroids formed hepatocyte-like colonies rather than cholangiocytes in vivo. Conclusion Our results indicated that the enhancement of hepatic functions in 3D-cultured HepaRG cells was induced by selective hepatocyte differentiation and accelerated hepatocyte maturation. HepaRG spheroids reproduced the metabolic responses of human hepatocytes. Therefore, FP-dependent 3D-cultured HepaRG cells may serve as an excellent in vitro model for evaluating the hepatic metabolism and toxicity. [ABSTRACT FROM AUTHOR]

1. Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient.2. A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83–86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat.3. Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting.4. By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1′-hydroxylation.5. These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6. [ABSTRACT FROM PUBLISHER]

1. The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies. 2. CYP2D, CYP2J2, CYP3A4 and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76 and CYP2E1 were not detected in any of the 35 animals examined. 3. On analysis of pooled microsomes (35 animals), CYP3A (3A4 + 3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%) and CYP2D (0.4%). On the analysis of individual microsome samples, each P450 content varied 2-to-6-fold between animals, and no sex differences were observed in any P450 content. 4. These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines. [ABSTRACT FROM AUTHOR]

1. Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology. [ABSTRACT FROM AUTHOR]

Monkeys, especially macaques, including cynomolgus ( Macaca fascicularis) and rhesus monkeys ( Macaca mulatta), are frequently used in drug metabolism studies due to their evolutionary closeness to humans. Recently, numerous cytochrome P450 (P450 or CYP) cDNAs have been identified and characterized in cynomolgus and rhesus monkeys and were named by the P450 Nomenclature Committee. However, recent advances in genome analysis of cynomolgus and rhesus monkeys revealed that some monkey P450s are apparently orthologous to human P450s and thus need to be renamed corresponding to their human orthologs. In this review, we focus on the P450s identified in cynomolgus and rhesus monkeys and present an overview of the identity and functional characteristics of each P450 cDNA in the CYP1-4 families. Information on the Japanese monkey ( Macaca fuscata), African green monkey ( Cercopithecus aethiops), and marmoset ( Callithrix jacchus), primate species used in some drug metabolism studies, are also included. We compared the genomic structure of the macaque P450 genes to those of human and rat P450 genes in the CYP1-4 families. Based on sequence identity, phylogeny, and genomic organization of monkey P450s, we determined orthologous relationships of monkey P450s and, in this article, propose a revised nomenclature: CYP2B17/CYP2B30 to CYP2B6, CYP2C20/CYP2C74 to CYP2C8, CYP2C43/CYP2C83 to CYP2C9, CYP2C75 to CYP2C19, CYP2F6 to CYP2F1, CYP3A8/CYP3A21/CYP3A64 to CYP3A4, CYP3A66 to CYP3A5, and CYP4F45 to CYP4F2. The information presented in this review is expected to promote a better understanding of monkey P450 genes through comparative genomics and thereby make it more feasible to use monkeys in drug metabolism studies. [ABSTRACT FROM AUTHOR]

Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84-86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1), an aberrantly spliced transcript (SV2) lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del). These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys), although its relative contribution to drug metabolism has yet to be validated. [ABSTRACT FROM AUTHOR]

1. A novel cytochrome P450 (CYP), CYP2A26, was identified and characterized in cynomolgus monkey, one of the animal species used in preclinical studies. 2. Deduced amino acid sequences of CYP2A26 cDNA showed high sequence identities (91-95%) with cynomolgus monkey CYP2A23 and CYP2A24, and human CYP2A6 and CYP2A13. 3. Phylogenetic analysis showed that macaque CYP2As (CYP2A26, CYP2A23, and CYP2A24) were most closely clustered with human CYP2As, unlike CYP2As of dog, rat, and mouse (other species also used in drug metabolism). 4. Quantitative polymerase chain reaction analysis showed that CYP2A26 mRNA, along with CYP2A23 and CYP2A24 mRNAs, was expressed predominantly in the liver, where CYP2A proteins were also detected by immunoblotting. 5. Drug-metabolizing assays using the CYP2A26 protein heterologously expressed in Escherichia coli indicated that CYP2A26 catalyzed coumarin 7-hydroxylation with its apparent Km lower than that of CYP2A24, but similar to those of CYP2A6 and CYP2A23. 6. These results suggest an evolutionary closeness and functional similarity of cynomolgus monkey CYP2A26 (together with CYP2A23 and CYP2A24) to human CYP2A6, and its functional role as a drug- metabolizing enzyme in the liver. [ABSTRACT FROM AUTHOR]

The article discusses the identification and characterization of drug-metabolizing enzyme cytochrome P450 2C18 (CYP2C18) in the cynomolgus macaque (Macaca fascicularis). The authors mention that they characterized the CYP2C18 cDNA in cynomolgus macaques. They explain that the cDNA was homologous to human CYP2C18 cDNA. They reveal that cynomolgus CYP2C18 was expressed in the liver and kidney. They conclude that the results indicate that cynomolgus CYP2C18 could act as a drug-metabolizing enzyme in the liver.

[Display omitted] Drug oxygenation is mainly mediated by cytochromes P450 (P450s, CYPs) and flavin-containing monooxygenases (FMOs). Polymorphic variants of P450s and FMOs are known to influence drug metabolism. Species differences exist in terms of drug metabolism and can be important when determining the contributions of individual enzymes. The success of research into drug-metabolizing enzymes and their impacts on drug discovery and development has been remarkable. Dogs and pigs are often used as preclinical animal models. This research update provides information on P450 and FMO enzymes in dogs and pigs and makes comparisons with their human enzymes. Newly identified dog CYP3A98, a testosterone 6β- and estradiol 16α-hydroxylase, is abundantly expressed in small intestine and is likely the major CYP3A enzyme in small intestine, whereas dog CYP3A12 is the major CYP3A enzyme in liver. The roles of recently identified dog CYP2J2 and pig CYP2J33/34/35 were investigated. FMOs have been characterized in humans and several other species including dogs and pigs. P450 and FMO family members have been characterized also in cynomolgus macaques and common marmosets. P450s have industrial applications and have been the focus of attention of many pharmaceutical companies. The techniques used to investigate the roles of P450/FMO enzymes in drug oxidation and clinical treatments have not yet reached maturity and require further development. The findings summarized here provide a foundation for understanding individual pharmacokinetic and toxicological results in dogs and pigs as preclinical models and will help to further support understanding of the molecular mechanisms of human P450/FMO functionality. [ABSTRACT FROM AUTHOR]

Cytosolic sulfotransferases (SULTs), drug-metabolizing enzymes essential for the metabolism of endogenous biochemicals and foreign compounds, have been characterized in humans, but remain to be investigated in cynomolgus macaques, important species in drug metabolism studies. In this study, based on the genome data, cynomolgus SULT1A1, SULT1A3, SULT1B1, SULT1C2v1, SULT1C2v2, SULT1C4, SULT1E1, and SULT2A1 cDNAs were isolated and characterized. Among these, cynomolgus SULT1C2v2 was highly homologous to human SULT1C2P1 (pseudogene). These cynomolgus SULT cDNAs had high sequence identities (95–97%) to, and closely clustered with their human orthologs in a phylogenetic tree. Gene structure and genomic organization of each cynomolgus SULT were similar to those of the human ortholog. Among the 10 tissue types analyzed, cynomolgus SULTs showed distinct expression patterns similar to human SULTs; more specifically, mRNA was most abundantly expressed in livers (SULT1A1, SULT1C2v2, SULT1C4, and SULT2A1), jejunum (SULT1A3, SULT1B1, and SULT1E1), or kidneys (SULT1C2v1). The most abundant SULT mRNA was SULT2A1, SULT1E1, and SULT1C4 found in livers, jejunum, and kidneys, respectively. Recombinant cynomolgus SULT1A1, SULT1A3, SULT1B1, SULT1C2v1, SULT1C2v2, SULT1C4, SULT1E1, and SULT2A1 in bacterial cytosolic fractions mediated 3′-phosphoadenosine-5′-phosphosulfate-dependent sulfate conjugations of typical human SULT substrates, 1-naphthol, p -nitrophenol, dopamine, dehydroepiandrosterone, and estradiol. Taken together, these results suggest molecular and functional similarities of SULTs between cynomolgus macaques and humans. [ABSTRACT FROM AUTHOR]

[Display omitted] Cytochromes P450 (P450s or CYPs), especially the CYP2C family, are important drug-metabolizing enzymes that play major roles in drug metabolism. Tree shrews, a non-rodent primate-like species, are used in various fields of biomedical research, notably hepatitis virus infection; however, its drug-metabolizing enzymes have not been fully investigated. In this study, tree shrew CYP2C18, CYP2C76a, CYP2C76b, and CYP2C76c cDNAs were identified and contained open reading frames of 489 or 490 amino acids with high sequence identities (70–78 %) to human CYP2Cs. Tree shrew CYP2C76a, CYP2C76b, and CYP2C76c showed higher sequence identities (79–80 %) to cynomolgus CYP2C76 and were not orthologous to any human CYP2C. Phylogenetic analysis revealed that tree shrew CYP2C18 and CYP2C76s were closely related to rat CYP2Cs and cynomolgus CYP2C76, respectively. Tree shrew CYP2C genes formed a gene cluster similar to human CYP2C genes. All four tree shrew CYP2C mRNAs showed predominant expressions in liver, among the tissue types examined; expression of CYP2C18 mRNA was also detected in small intestine. In liver, CYP2C18 mRNA was the most abundant among the tree shrew CYP2C mRNAs. In metabolic assays using human CYP2C substrates, all tree shrew CYP2Cs showed metabolic activities toward diclofenac, R,S -omeprazole, paclitaxel, and R,S -warfarin, with the activity of CYP2C18 exceeding that of the other CYP2Cs. Moreover, tree shrew CYP2C76 enzymes metabolized progesterone more efficiently than human, cynomolgus, or marmoset CYP2Cs. Therefore, these novel tree shrew CYP2Cs are expressed abundantly in liver, encode functional enzymes that metabolize human CYP2C substrates, and are likely responsible for drug clearances. [ABSTRACT FROM AUTHOR]

Abstract: Flavin-containing monooxygenases (FMOs), drug-metabolizing enzymes essential for the metabolism of endogenous biochemicals and foreign compounds, have been characterized in human (including FMO1-5 and FMO6P), but remain to be investigated in cynomolgus macaque. In this study, cDNAs of cynomolgus FMO1-5 and FMO6 were isolated and characterized. Amino acid sequences of cynomolgus FMO1-5, respectively, shared high sequence identities (94–98%) and were closely clustered in a phylogenetic tree, with human FMO1-5. Eight different transcripts, due to alternative splicing, were isolated for cynomolgus FMO6, which is highly identical (~96%) to human FMO6P. Among the 10 tissue types analyzed, cynomolgus FMO1, FMO2, FMO4, and FMO6 were most abundantly expressed in kidney, while cynomolgus FMO3 and FMO5 were most abundantly expressed in liver. In kidney and liver, the most abundantly expressed cynomolgus FMO genes were FMO1 and FMO3, respectively. Cynomolgus FMO1, FMO2, FMO3, and FMO5 metabolized benzydamine, and FMO1/FMO3 and FMO3 also metabolized methimazole and trimethylamine, respectively. Rates of benzydamine N-oxygenation (catalyzed by FMO3) varied (approximately 20-fold) among the 28 cynomolgus livers and were significantly correlated with FMO3 protein expression, indicating that the inter-animal variations in benzydamine N-oxygenation might be partly accounted for by the variable FMO3 expression. Cynomolgus FMO6 metabolized benzydamine only slightly, but minimal expression of FMO6 in all tissue precludes the importance of FMO6 in drug metabolism, unlike cynomolgus FMO1, FMO2, FMO3, and FMO5 which were all functional. Abundant expression of FMO1 and FMO3 in kidney and liver, respectively, suggest their importance in drug metabolism in cynomolgus macaque, similar to human. [Copyright &y& Elsevier]

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1–5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1–6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85–90 % and 81–89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1–6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N -oxygenations and methyl p -tolyl sulfide S -oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1–6, which are orthologous to human FMOs (FMO1–5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver. [Display omitted] • Orthologs of human FMOs (FMO1–5 and FMO6P) were isolated from tree shrews. • Tree shrew FMO3 has molecular and functional characteristics similar to those of human FMO3. • FMO6 is an active form similar to FMO3, unlike the human pseudogene FMO6P. [ABSTRACT FROM AUTHOR]

In humans, polymorphic N -acetyltransferases NAT1 and NAT2 are important enzymes that metabolize endogenous and exogenous compounds, including drugs. These enzymes exhibit considerable inter-individual variability in humans. The cynomolgus macaque is a nonhuman primate species that is widely used in drug metabolism studies. NAT1/2 in these macaques have molecular and enzymatic similarities to their human orthologs; however, genetic polymorphisms in NAT1/2 have not been fully investigated in this species. In this study, the resequencing of NAT1 and NAT2 in 114 cynomolgus macaques and 19 rhesus macaques found 15 non-synonymous variants for NAT1 and 11 non-synonymous variants and 1 insertion/deletion variant for NAT2. Nine (60%) and five (33%) NAT1 variants and seven (67%) and three (25%) NAT2 variants were unique to cynomolgus and rhesus macaques, respectively. Functional characterization of the mutant enzymes was carried out using cynomolgus NAT1 and NAT2 proteins heterologously expressed in Escherichia coli. Compared with wild-type NAT1, the D122N NAT1 variant showed substantially lower acetylation activities toward p -aminobenzoic acid but had higher acetylation activities toward isoniazid. Moreover, liver cytosolic fractions from cynomolgus macaques homozygous for T98A NAT2 showed significantly lower acetylation activities toward isoniazid than wild-type NAT2; similar results were obtained for recombinant T98A NAT2. Interestingly, all the rhesus macaques analyzed were homozygous for T98A. These findings indicate that polymorphic NAT1/2 variants in cynomolgus and rhesus macaques, especially the T98A NAT2 variant, could account for the inter-animal and/or inter-lineage variabilities of NAT-dependent drug metabolism in macaques. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract UDP-glucuronosyltransferases (UGTs) are essential enzymes metabolizing endogenous and exogenous chemicals. However, characteristics of UGTs have not been fully investigated in molecular levels of cynomolgus macaques, one of non-human primates widely used in preclinical drug metabolism studies. In this study, three UGT2A cDNAs (UGT2A1, 2A2, and 2A3) were isolated and characterized along with seven UGT2Bs previously identified in cynomolgus macaques. Several transcript variants were found in cynomolgus UGT2A1 and UGT2A2, like human orthologs. Cynomolgus UGT2A and UGT2B amino acid sequences were highly identical (87–96%) to their human counterparts. By phylogenetic analysis, all these cynomolgus UGT2s were more closely clustered with their human homologs than with dog, rat, or mouse UGT2s. Especially, UGT2As showed orthologous relationships between humans and cynomolgus macaques. All the cynomolgus UGT2 mRNAs were expressed in livers, jejunum, and/or kidneys abundantly, except that UGT2A1 and UGT2A2 mRNAs were predominantly expressed in nasal mucosa, like human UGT2s. UGT2A and UGT2B genes together form a gene cluster in the cynomolgus and human genome. Among the seven cynomolgus UGT2Bs heterologously expressed in yeast, UGT2B9 and UGT2B30 showed activities in estradiol 17- O -glucuronidation and morphine 3- O -glucuronidation but did not show activities in estradiol 3- O -glucuronidation, similar to human UGT2Bs. In liver microsomes, cynomolgus macaques showed higher estradiol 17- O -glucuronidase and morphine 3- O -glucuronidase activities than humans, suggesting functional activities of the responsible UGT2B enzymes in cynomolgus macaques. Therefore, cynomolgus UGT2s had overall molecular similarities to human UGT2s, but also showed some differences in UGT2B enzyme properties. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. Molecular characteristics of UGTs have been extensively investigated in humans, but in cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, remain to be investigated. In this study, 12 UGT1A cDNAs (UGT1A1, 1A2, 1A4A, 1A4B, 1A5A, 1A5B, 1A5C, 1A6, 1A7, 1A8, 1A9, and 1A10) were isolated and characterized in cynomolgus macaques. UGT1A5C cDNA did not contain a complete coding region due to nonsense mutations, and was excluded from further analysis. Amino acid sequences of all 11 cynomolgus UGT1As had high sequence identities (92–95%) with human UGT1As and were phylogenetically close to human UGT1As. These cynomolgus UGT1A genes shared exons 2–5, and contained a variable exon 1 unique to each gene, similar to human UGT1A genes. Moreover, cynomolgus and human UGT1A gene clusters were located in corresponding regions in the genome. Among the 10 tissue types analyzed, cynomolgus UGT1A mRNAs were most abundantly expressed in the liver, jejunum, and/or kidney, the drug-metabolizing organs, similar to human UGT1As. Among these 11 cynomolgus UGT1A mRNAs, cynomolgus UGT1A2, UGT1A9, and UGT1A10 mRNAs were most abundantly expressed in the liver, kidney, and jejunum, respectively. Cynomolgus liver microsomes and UGT1A proteins catalyzed glucuronidation of the substrates human UGT1As catalyze, including 4-methylumbelliferone, 4-nitrophenol, estradiol, trifluoperazine, serotonin, and propofol, although trifluoperazine glucuronidation was not catalyzed by any cynomolgus UGT1A proteins. These results suggest that cynomolgus UGT1As are functional enzymes with molecular similarities to human UGT1As. [ABSTRACT FROM AUTHOR]

CYP2C19 (formerly known as CYP2C75), highly homologous to human CYP2C19, has been identified in cynomolgus and rhesus macaques, non-human primate species widely used in drug metabolism studies. CYP2C19 is predominantly expressed in liver and encodes a functional drug-metabolizing enzyme. Genetic variants in human CYP2C genes account for the inter-individual variability in drug metabolism; however, genetic variants have not been investigated in macaque CYP2C19 . In the present study, re-sequencing of CYP2C19 in 78 cynomolgus and 36 rhesus macaques identified 34 non-synonymous variants. Among these, 6 were located in substrate recognition sites, the domains important for protein function. Eighteen and 6 variants were unique to cynomolgus and rhesus macaques, respectively. Four variants were characterized by site-directed mutagenesis and metabolic assays, and 3 variants (p.Phe100Asn, p.Ala103Val, and p.Ile112Leu) showed substantially reduced activity as compared with wild type in flurbiprofen 4′-hydroxylation, omeprazole 5-hydroxylation, and R- / S -warfarin 7-hydroxylation. These variants, co-segregating in the animals analyzed, influenced metabolic activities because the homozygotes and/or heterozygotes showed significantly reduced catalytic activities in liver toward flurbiprofen 4′-hydroxylation and omeprazole 5-hydroxylation as compared with wild type. Kinetic analysis for R -warfarin 7-hydroxylation and docking simulation indicated that CYP2C19 Ala103Val would change the function and conformation of this enzyme. Ala103Val variation diminished homotropic cooperativity of CYP2C19 with R -warfarin yielding low metabolic capacity. These results indicated that the interindividual variability of CYP2C-dependent drug metabolism is at least partly accounted for by CYP2C19 variants in cynomolgus macaques. [ABSTRACT FROM AUTHOR]

Abstract: Glutathione S-transferases (GSTs) are essential drug-metabolizing enzymes, involved in conjugation of various endogenous and exogenous substrates. Cynomolgus macaque is an important primate species in drug metabolism studies; however, cynomolgus GSTs have not been fully characterized. In this study the cDNAs of 12 GSTs (GSTA3-A5, GSTK1, GSTM2-M4, GSTO2, GSTP1, GSTS1, and GSTT1/2) were isolated from cynomolgus macaque and rhesus macaque liver. Cynomolgus GSTM1 cDNA was not amplified and only an aberrantly spliced GSTM1 transcript was isolated from rhesus macaque. Amino acid sequences of these 12 GSTs shared high sequence identities (93–98%) and were clustered into the same clades as the human orthologs in the phylogenetic tree. The 12 GSTs had exon–intron structures similar to the human orthologs, and exhibited distinct tissue expression patterns. GSTA3, GSTA5, and GSTM3/O2 were expressed predominantly in adrenal gland, jejunum, and testis, respectively, whereas the other GSTs showed universal expression patterns in the 10 tissues analyzed. Comparison of expression levels showed that GSTA1, GSTK1, GSTA3, and GSTM3 were most abundantly expressed in liver/jejunum, kidney, adrenal gland, and testis, respectively. Metabolic assays of proteins expressed heterologously in Escherichia coli, showed that all 12 GSTs and 5 previously identified GSTs, GSTA1/2, GSTM5, GSTO1, and GSTZ1, catalyzed the conjugation of GST substrate(s) 1-chloro-2,4-dinitrobenzene and/or 1,2-epoxy-3-(p-nitrophenoxy)propane, indicating that these 17 GSTs are functional drug-metabolizing enzymes. These results suggest that the 12 GST genes examined in this study are expressed and encoded functional enzymes in cynomolgus macaque. [Copyright &y& Elsevier]

Abstract: Cytochrome P450 (P450 or CYP) 1 family consists of the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. In the human genome, CYP1A1, CYP1A2, and CYP1B1 are expressed and encode functional enzymes, whereas CYP1D1P (formerly known as CYP1A8P) is present as a pseudogene due to five nonsense mutations in the putative coding region. In this study, we identified CYP1D1 cDNA, highly identical (nearly 95%) to human CYP1D1P sequence, in cynomolgus monkey, a species frequently used in drug metabolism studies due to its evolutionary closeness to human. The amino acid sequence deduced from cynomolgus monkey CYP1D1 cDNA shared the high sequence identity (91%) with human CYP1D1P (postulated from the gene sequence), and the highest sequence identity (44–45%) with CYP1A1 and CYP1A2 among cynomolgus monkey P450s. CYP1D1 mRNA was most abundantly expressed in liver, followed by kidney, and jejunum. The hepatic expression level of CYP1D1 mRNA was comparable to that of CYP1A1 mRNA and much higher than that of CYP1A2 mRNA. CYP1D1 was barely detectable in immunoblots of cynomolgus monkey liver. Cynomolgus monkey CYP1D1 mRNA was induced in primary hepatocytes with omeprazole. Cynomolgus monkey CYP1D1 protein heterologously expressed in Escherichia coli catalyzed ethoxyresorufin O-deethylation and caffeine 8-hydroxylation, which CYP1As also catalyze. Finally, no nonsense mutations, corresponding to those found in human CYP1D1P, were found in the 20 cynomolgus monkeys and 10 rhesus monkeys used in this study. These results suggest that CYP1D1 plays a role as a functional, drug-metabolizing enzyme in cynomolgus monkey liver. [ABSTRACT FROM AUTHOR]

Abstract: Background: Genetic polymorphisms of the human CYP2D6 gene can affect the metabolism of many drugs in clinical use. As a first step toward identifying poor drug metabolizers in the clinical setting, we developed a new multiplex PCR-based genotyping method to detect CYP2D6 whole-gene deletion. Methods: We validated the new method by analyzing 500 genomic DNA samples from a Japanese population with the conventional long-PCR method and the new multiplex PCR method. The long-PCR system used a forward primer for CYP2D7P (a pseudogene closely related to CYP2D6) and a common reverse primer for the untranslated region. The multiplex PCR system used the same two primers as the long PCR and an additional forward primer for CYP2D6. Results: With the long-PCR system, DNA samples identified as containing CYP2D6*5 (whole-gene deletion) formed 3.5-kb PCR products. With the multiplex PCR system, many samples yielded 4.7-kb PCR products (implying the existence of normal CYP2D6) and some DNA samples yielded 6.2-kb PCR products (probably indicating CYP2D6*10D). The long-PCR assay detected 64 CYP2D6*5 alleles among 1000 Japanese alleles; however, the new multiplex PCR system identified 5 of these 64 alleles as CYP2D6*10D. Conclusions: The new multiplex PCR method is useful for detecting CYP2D6*5. This system could reliably discriminate CYP2D6*5 from homologous pseudogene CYP2D7P and functional CYP2D6*10D. [Copyright &y& Elsevier]

Cytosolic sulfotransferases (SULTs), which mediate the conjugation of drugs with 3′-phosphoadenosine-5′-phosphosulfate, have been characterized in humans and cynomolgus monkeys. However, SULTs remain to be evaluated in common marmosets, a species of non-human primate often employed in drug metabolism and pharmacokinetic studies of endogenous and exogenous compounds. In this study, marmoset SULT1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 cDNAs were isolated and characterized, based on genome data. The deduced amino acid sequences of these marmoset SULT cDNAs had high identities (90–95%) with their human orthologs, except for marmoset SULT2A1, which was only 81% identical to human SULT2A1. The amino acid sequences of the orthologs of these six SULTs in marmosets, monkeys, and humans were closely clustered in a phylogenetic tree. The structures and genomic organizations of marmoset SULT genes were similar to those of their human orthologs. Among the five marmoset tissues analyzed, SULT mRNAs showed typical expression patterns. The most abundant SULT mRNAs were SULT1B1 in liver, small intestine, and kidney; SULT1E1 in lung; and SULT1A3 in brain. Recombinant marmoset SULT1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 proteins expressed in bacterial cytosolic fractions mediated sulfate conjugations with 3′-phosphoadenosine-5′-phosphosulfate of the following typical human SULT substrates: dopamine, 1-naphthol, p -nitrophenol, estradiol, and dehydroepiandrosterone. Taken together, these wide-ranging results suggest functional and molecular similarities of SULTs among marmosets, monkeys, and humans. [ABSTRACT FROM AUTHOR]

The common marmoset is an important primate species used in drug metabolism studies. However, glutathione S -transferases (GSTs), essential drug-metabolizing enzymes involved in the conjugation of various endogenous and exogenous substrates, have not been identified or characterized in this species. In this study, 20 GSTs [including 3 microsomal GSTs (MGSTs)] were identified and characterized in marmosets. Marmoset GSTs had amino acid sequences highly identical (86–99%) to human GSTs, except for GSTA4L, which had lower identities (59–62%) with human GSTAs. Phylogenetic analysis revealed that marmoset GSTs were closely clustered with their human counterparts. Marmoset GST s had gene and genomic structures generally similar to their human counterparts, with some differences in GSTA , GSTM , and GSTT clusters. Marmoset GST mRNAs exhibited distinct tissue expression patterns: GSTA1, GSTA3, GSTA4L, GSTK1, GSTT1, GSTZ1, and MGST1 mRNAs were expressed most abundantly in liver. Other GST mRNAs were expressed most abundantly in small intestine, lung, brain, or kidney. Expression of GSTT4 and GSTT4L mRNAs was detected only in testis. Among all 20 marmoset GST mRNAs, the most abundant mRNAs were GSTA1 mRNA in liver, small intestine, and kidney; GSTM3 mRNA in testis; and MSGT3 mRNA in brain and lung. All 20 GSTs mediated the conjugation of GST substrates 1-chloro-2,4-dinitrobenzene; 1,2-epoxy-3 - (p-nitrophenoxy)propane; styrene 7,8-oxide; and/or 1-iodohexane, but with different activity levels. Kinetic analyses showed that marmoset GSTM2/GSTM5 and GSTM5/GSTT1 effectively conjugated styrene 7,8-oxide and 1-iodohexane, respectively, with the highest affinity. These results suggest that the 20 newly identified marmoset GSTs were functional drug-metabolizing enzymes able to conjugate typical GST substrates. [ABSTRACT FROM AUTHOR]

UDP-glucuronosyltransferases (UGTs) are essential drug-conjugation enzymes that metabolize a variety of endobiotic and xenobiotic substrates. The molecular characteristics of UGTs have been extensively investigated in humans, but remain to be investigated in common marmosets, a nonhuman primate species widely used in drug metabolism studies. In this study, 11 UGT cDNAs (UGT1A1, 1A3, 1A4, 1A6, 1A7, and 1A9; and UGT2B49, 2B50, 2B51, 2B52, and 2B53) were isolated and characterized in marmosets. Marmoset UGT1As had high sequence identities (89–93%) with human UGT1As, but the sequence identities of marmoset UGT2Bs were lower (82–86%). Marmoset UGTs were found to be phylogenetically close to human UGTs. Just as human UGT1As do, marmoset UGT1A genes shared exons 2–5 and contained a variable exon 1 unique to each gene; in contrast, marmoset UGT2B genes contained six unique exons. Moreover, marmoset and human UGT1A and UGT2B gene clusters were located in corresponding regions in their respective genomes. Among the five tissue types tested, marmoset UGT mRNAs were most abundantly expressed in liver, jejunum, and/or kidney, i.e., in tissues important for drug metabolism, just as human UGTs are. Among the 11 marmoset UGT mRNAs investigated, marmoset UGT1A9, 1A4, and 1A6 mRNAs were the most abundantly expressed in liver, small intestine, and kidney, respectively. Marmoset liver microsomes and recombinant UGT1A proteins catalyzed the glucuronidation of the same substrates that human UGT1As catalyze, including estradiol, trifluoperazine, 4-methylumbelliferone, serotonin, 4-nitrophenol, and propofol. Trifluoperazine was glucuronidated by marmoset liver microsomes, but not by any of the UGT1A isoforms examined under the present conditions. These results collectively suggest that functional marmoset UGTs have generally similar molecular characteristics to human UGTs. [ABSTRACT FROM AUTHOR]