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Background: Rapid identification of causative bacteria in treatment of acute otitis media (AOM) is of paramount importance for appropriate antibiotic use., Materials and Methods: This prospective observational study was conducted in 15 hospitals and clinics in Japan between 2018 and 2020. A new rapid antigen test kit (AOS-116), which simultaneously detects antigens for Streptococcus pneumoniae (Sp) and Haemophilus influenzae (Hi), was applied for middle ear fluids (MEFs) and nasopharyngeal secretions (NPSs) in patients with moderate to severe AOM. We investigated relationship between the results of rapid test, severity at initial visit, and clinical course., Results: Regarding performance accuracy based on culture results, AOS-116 showed 1) high (>80%) sensitivity, specificity, and negative predictive value (NPV) in MEFs for both antigens, 2) high sensitivity, specificity, and positive predictive value (PPV) in NPSs for Hi antigen, and 3) high specificity, and PPV in NPSs for Sp antigen. Regarding predictive value of nasopharyngeal culture and antigen detection for causative middle ear pathogens, similar results were observed between AOS-116 and culture, which was characterized with high sensitivity and NPV for both pathogens. MEFs/NPSs positive for Hi antigen were significantly associated with eardrum findings, and severity. MEFs/NPSs positive for pneumococcal antigen were significantly associated with severity of otalgia, fever, and otorrhea. Among patients with prior antimicrobial treatment, improvement tended to be slower in cases positive for Hi than in cases negative., Conclusion: The rapid antigen detection test is useful as a decision-making tool for prescribing antimicrobial agents and may play an important role in promoting appropriate antimicrobial use., Competing Interests: Declaration of competing interest The authors declare financial support from ASAHI KASEI Corporation. Takuji Miyata and Norikazu Okamura are employees of Asahi Kasei Corporation. The funder had no role in the experimental design, implementation of the study, interpretation of the data, or decision to submit the manuscript for publication. This does not alter the authors’ adherence to any publication policies on sharing data and materials., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Human induced pluripotent stem (hiPS) cells have demonstrated promising potential in regenerative medical therapeutics. After successful clinical trials, the demand for hiPS cells has steadily increased. Therefore, the optimization of hiPS cell freezing processes for storage and transportation is essential. Here, we presented a computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for hiPS cells. This study was based on a model that calculates cell survival rates after thawing, and the model was extended to evaluate cell potentials until 24 h after seeding. To estimate parameter values for this extension, freezing experiments were performed using constant cooling rates. Using quality and productivity indicators, we evaluated 16,206 temperature profiles using our model, and a promising profile was obtained. Finally, an experimental investigation of the profile was undertaken, and the contribution of the temperature profile to both quality and productivity was confirmed., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Drug-metabolizing enzymes are important in drug development and therapy, but have not been fully identified and characterized in many species, lines, and breeds. Liver transcriptomic data were analyzed for phase I cytochromes P450, flavin-containing monooxygenases, and carboxylesterases and phase II UDP-glucuronosyltransferases, sulfotransferases, and glutathione S-transferases. Comparisons with a variety of species (humans, rhesus macaques, African green monkeys, baboons, common marmosets, cattle, sheep, pigs, cats, dogs, rabbits, tree shrews, rats, mice, and chickens) revealed both general similarities and differences in the transcript abundances of drug-metabolizing enzymes. Similarly, Beagle and Shiba dogs were examined by next-generation sequencing (RNA-seq). Consequently, no substantial differences in transcript abundance were noted in different breeds of pigs and dogs and in different lines of mice and rats. Therefore, the expression profiles of hepatic drug-metabolizing enzyme transcripts appear to be similar in Shiba and Beagle dogs and pig breeds and the rat and mouse lines analyzed, although some differences were found in other species., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (© 2024 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The response of basilar arteries to noradrenaline varies among many animal species, but remains little studied in poultry. Accordingly, we aimed to characterize the adrenergic receptor (AR) subtypes that modulate vascular response in basilar arteries in the chicken, with isometric recording of arterial ring tension using an organ bath. We demonstrated the presence of both alpha and beta (α and β) receptor subtypes through evaluating the response to noradrenaline, with and without a range of β-AR and α-AR antagonists. The concentration-dependent relaxations then induced by a range of β-AR agonists indicated a potency ranking of isoproterenol > noradrenaline > adrenaline > procaterol. We then investigated the effects of β-AR antagonists that attenuate the effect of isoproterenol (propranolol for β 1,2,3 -ARs, atenolol for β 1 -ARs, butoxamine for β 2 -ARs, and SR 59230A for β 3 -ARs), with Schild regression analysis, ascertaining multiple β-AR subtypes, with neither the β 1 -AR nor the β 2 -AR as the dominant subtype. SR 59230A was the only antagonist to yield a pA 2 value (7.52) close to the reported equivalent for the relevant receptor subtype. Furthermore, treatment with SR 58611 (a β 3 -AR agonist) induced relaxation, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Additionally, treating basilar arterial strips (containing endothelium) with SR 58611 induced nitric oxide (NO) production, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Based on this first characterization of AR subtypes in chicken basilar arteries (to our knowledge), we suggest that α- and β-ARs are involved in contraction and relaxation, and that β 3 -ARs, especially those on the endothelium, may play an important role in vasodilation via NO release., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Cynomolgus macaques are used in preclinical studies in part because of their evolutionary closeness to humans. However, drug transporters [including solute carrier (SLC) transporters] essential for the absorption and excretion of drugs have not been fully investigated at the molecular level in cynomolgus macaques. We identified and characterized cynomolgus macaque SLC15A1, SLC15A2, SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLC47A1, and SLC47A2, along with SLCO (formerly SLC21A) transporters SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1. These cynomolgus SLC transporters had high amino acid sequence identities (92-97%) with their human orthologs and contained sequence motifs characteristic of SLC transporters. Phylogenetic analysis showed that these cynomolgus SLC transporters were more closely clustered with their human orthologs than with those of dogs, rats, or mice. Gene structure and genomic organization were similar in macaques and humans. Cynomolgus SLC transporter mRNAs showed distinct tissue expression patterns, being most abundantly expressed in jejunum (SLC15A1), liver (SLC22A1, SLCO1B1, and SLCO2B1), and kidney (SLC15A2, SLC22A2, SLC22A6, SLC22A8, SLC47A1, SLC47A2, and SLCO1A2). In contrast, cynomolgus SLCO2B1 mRNA was more ubiquitously expressed. Among these SLC mRNAs, the most abundant in liver was SLCO1B1, in jejunum SLC15A1, and in kidney SLC22A2. These results suggest similar characteristics of SLC transporters in cynomolgus macaques and humans., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Cynomolgus macaques are used in preclinical studies in part because of their evolutionary closeness to humans. However, drug transporters, including ATP-binding cassette (ABC) transporters, which are essential for the absorption and excretion of drugs, have not been fully investigated at the molecular level in cynomolgus macaques. In this study, ABCB4, ABCC3, ABCC4, and ABCG2 cDNAs were newly identified and characterized, along with ABCB1, ABCB11, and ABCC2 cDNAs previously identified, in cynomolgus macaques. All seven cynomolgus ABC transporters had high sequence identities (96-98%) with their human orthologs in terms of amino acid sequences and were also most closely clustered with their human orthologs by phylogenetic analysis. Furthermore, the gene structures and genomic organization were similar in cynomolgus macaques and humans. The mRNAs of these cynomolgus ABC transporters, as analyzed using the quantitative polymerase chain reaction, showed distinct tissue expression patterns. Among the ten tissues, ABCB1, ABCC2, ABCC3, and ABCG2 mRNAs were most abundantly expressed in jejunum; ABCB4 and ABCB11 in liver; and ABCC4 in kidney, which are similar to the expression patterns of human ABC transporters. These results suggest molecular similarities of the ABC transporters in cynomolgus macaques and humans., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Paraoxonase (PON) plays roles in the metabolism of organophosphate xenobiotics and drugs. Despite the importance of marmosets for research into drug metabolism and pharmacokinetics, marmoset paraoxonase has not yet been fully characterized. Consequently, we identified the PON1 gene in the marmoset genome by sequence homology analysis. Marmoset PON1 cDNA containing an open reading frame (1065 bp) was successfully cloned from marmoset liver by reverse transcription-polymerase chain reaction. The deduced amino acid sequence (355 amino acids) has approximately 93% identity with the human ortholog and contains important amino acid residues for substrate binding and calcium ion coordination. According to a phylogenetic tree of PON1 amino acid sequences constructed using data from seven animal species, marmoset PON1 is closer to human PON1 than it is to the PON1 orthologs of experimental animals such as pigs, rabbits, rats, and mice. Marmoset PON1 mRNA was predominantly expressed in liver among the five tissues examined. Marmoset PON1 protein secreted into plasma was detected by immunoblotting. The paraoxon-hydrolyzing activity in plasma was higher in marmosets than in humans. Based on these data, we concluded that marmoset and human PON1 have similar characteristics with regard to genomic structure, amino acid sequences, and tissue distribution., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The biodistribution profile of cell-based therapy products in animal models is important for evaluation of their safety and efficacy. Because of its quantitative nature and sensitivity, the quantitative polymerase chain reaction (qPCR) is a useful method for detecting and quantifying xenogeneic cell-derived DNA in animal models, thereby allowing a biodistribution profile to be established. Although the restriction endonuclease family from Arthrobacter luteus (Alu) of repetitive elements in human genome sequences has been used to assess the biodistribution of human cells, high background signals are detected. In the present study, we evaluate the potential of domain of unknown function 1220 (DUF1220), which is a human lineage-specific, multiple-copy gene similar to Alu sequences, for such analysis. Using qPCR analysis for DUF1220, human genome could be detected against a mouse genome background at a level comparable to that of Alu sequences with no detectable background signals. Moreover, using this approach, the human genome could be distinguished from the cynomolgus monkey genome. Further investigation of the quantitative aspects of this DUF1220-based qPCR assay might prove its usefulness for biodistribution studies of human cells transplanted into animals in the future., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Objectives: To facilitate better antibiotic stewardship, we conducted this clinical trial to identify the prognostic features of treatment failure in pediatric acute otitis media (AOM)., Study: Design: This is a randomized, parallel-group, open-label, comparative clinical trial., Subjects and Methods: Children with AOM and aged between 1 month and 5 years were enrolled. Patients were randomly assigned to receive either amoxicillin alone (70 mg/kg) for five days, or the same with additional clarithromycin (15 mg/kg) for the initial three days. The clinical course of AOM was evaluated based on tympanic membrane scores. Failure of treatment for AOM was confirmed on day 14. Nasal conditions were also assessed by a clinical scoring system for acute rhinosinusitis., Results: Treatment failures occurred in 25 out of 129 (19.4%) children. The ratio of treatment failures by age was significantly higher in children younger than 2 years than in children older than 2 years. The tympanic membrane scores on day 3 (P = 0.0334) and day 5 (P < 0.0001) and acute rhinosinusitis scores on day 5 (P = 0.0004) were higher in failure cases than in cured cases. Multivariate logistic regression analysis indicated significant associations between the treatment failure with tympanic membrane scores and acute rhinosinusitis scores on day 5, and the antimicrobial treatment regimen., Conclusions: Improvement of acute rhinosinusitis and tympanic membrane scores on day five were important predictive features in failure of treatment for pediatric AOM. These results will be useful when discussing the treatment decisions with the patient's parents., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Polymorphic human and cynomolgus macaque flavin-containing monooxygenases (FMO) 3 are important oxygenation enzymes for nitrogen-containing drugs. Inter-animal variability of FMO3-dependent drug oxygenations in vivo is suspected in cynomolgus macaques because such variability is evident in humans. Therefore, this follow-up study was performed to investigate the pharmacokinetics of orally administered deuterium-labeled trimethylamine in three cynomolgus macaques genotyped for FMO3. Trimethylamine-d 9 was rapidly absorbed and attained plasma concentrations greater than the background levels of non-labeled trimethylamine. Trimethylamine-d 9 was then converted to trimethylamine-d 9 N-oxide. The half-lives, maximum plasma concentrations, and areas under the curve for trimethylamine-d 9 and its N-oxygenated metabolite and the total clearance for orally administered trimethylamine-d 9 were not different among the heterozygote for Q506K FMO3, the heterozygote for V325I FMO3, and the heterozygote for both S99N and F510S FMO3. Trimethylamine N-oxygenation activities mediated by liver microsomes prepared from the same three animals were not substantially different. However, recombinant proteins of the corresponding cynomolgus FMO3 variants showed apparent reduced trimethylamine N-oxygenation activities compared with the wild-type proteins. This study suggests only limited polymorphic effects on the in vivo catalytic function of cynomolgus FMO3. These findings yield important insights in terms of both quantitative and qualitative variations of polymorphic FMO3 in cynomolgus liver., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The common marmoset (Callithrix jacchus) is a New World monkey that is increasingly used in pharmacological and toxicological studies. Thiopurine S-methyltransferase (TPMT) plays roles in the metabolism of widely used anticancer and anti-inflammatory drugs. Here, we report the isolation and molecular characterization of marmoset TPMT cDNA, which was found to contain an open-reading frame of 245 amino acids that was approximately 92% identical to its human ortholog. Marmoset TPMT was phylogenetically closer to other primate orthologs than to its pig, dog, rabbit, or rodent orthologs. Among the five marmoset tissue types analyzed, marmoset TPMT mRNA was most abundant in kidney and liver, just as human TPMT is. These results suggest that marmoset and human TPMT are similar at the molecular level., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The common marmoset (Callithrix jacchus), a New World primate, is currently attracting much attention as a nonhuman primate model for pharmacological and pharmacokinetic studies in preclinical research. In this study, we newly isolated the cDNAs of marmoset monoamine oxidase A (MAO-A) and MAO-B from liver and brain, respectively. MAO-A and MAO-B cDNAs, respectively, contained open reading frames of 527 and 520 amino acids and were approximately 92% and 95% identical to their human orthologs. Marmoset MAOs were phylogenetically closer to primate MAOs, including human MAOs, than to pig, dog, or rodent MAOs. The genomic and gene structures of marmoset MAOs were similar to those of humans. Among the five marmoset tissue types analyzed, the expression levels of MAO-A mRNA were relatively abundant in lung, liver, kidney, and small intestine, whereas the expression levels of MAO-B mRNA were relatively abundant in brain, liver, kidney, and small intestine; these tissue distributions are similar to those of human MAOs. These results suggest that MAO-A and MAO-B are similar at a molecular level in marmosets and humans., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The cynomolgus macaque is a nonhuman primate species that is often used in drug metabolism studies during drug development. However, the localization of UDP-glucuronosyltransferases (UGTs), essential drug-metabolizing enzymes, has not been fully investigated in the liver and small intestine of cynomolgus macaques. In this study, UGT activities were analyzed in liver (five lobes) and small intestine (the duodenum and six sections from the proximal jejunum to the distal ileum) using typical probe substrates of human UGTs: 7-hydroxycoumarin, estradiol, serotonin, propofol, and zidovudine. In liver, UGT activities with respect to all substrates were detected, and the activity levels were similar in all liver lobes of the cynomolgus macaques tested. In contrast, in the small intestine, UGT activities toward all substrates were detected, but their levels generally decreased from jejunum to ileum in cynomolgus macaques. The localization of estradiol 3-O-glucuronosyltransferases and serotonin O-glucuronosyltransferases (which are mainly UGT1A enzymes) appear to be different in liver and small intestine. These results collectively suggest that, in cynomolgus macaques, UGT1As are differentially localized in the small intestine but are relatively homogeneously distributed in the liver., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. The cynomolgus macaque is a nonhuman primate species widely used in drug metabolism studies. The molecular characteristics of UGTs have been extensively investigated in humans, but they remain to be elucidated in cynomolgus macaques. In this study, cynomolgus macaque UGT3A1, UGT3A2, and UGT8A1 cDNAs were isolated and characterized. Amino acid sequences deduced from cynomolgus UGT3A1, UGT3A2, and UGT8A1 cDNAs were highly identical with their human orthologs (93, 96, and 99%, respectively) and were closely clustered in a phylogenetic tree. In the genome, cynomolgus UGT3A and UGT8A genes were located in the regions corresponding to those of their human orthologs. Among the 10 tissue types analyzed, expression of cynomolgus UGT3A1 and UGT3A2 mRNAs was detected in liver, kidney, and testis; the UGT3A1 and UGT3A2 mRNAs were most abundant in liver and testis, respectively. Cynomolgus UGT8A1 was most abundantly expressed in kidney, followed by brain, jejunum, and testis. These results suggest that cynomolgus UGT3As and UGT8A1 have molecular similarities to their human orthologs., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The cynomolgus macaque, partly due to its evolutionary closeness to humans, is an important nonhuman primate species used in drug metabolism studies. In humans, expressions of cytochromes P450 (P450s), including the important drug-metabolizing enzyme P450 3A4, are affected by various cytokines. However, this phenomenon has not been fully investigated in cynomolgus macaques. In this study, the effects of cytokines on P450 expression were investigated using the quantitative polymerase chain reaction to evaluate mRNA expression. Hepatocytes from cynomolgus macaques were treated with lipopolysaccharide and various cytokines, including interleukin (IL)-1β, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α, and the expression levels of 11 P450s were compared with those of solvent-treated controls. Tumor necrosis factor-α significantly decreased cynomolgus P450 2C8 and 2C76 mRNA expression in multiple lots of cynomolgus hepatocytes investigated. IL-1β significantly decreased cynomolgus P450 1A1, 2C8, 2C19, and 2C76 mRNA expression, but increased P450 3A5 mRNA expression in multiple lots of hepatocytes. Moreover, P450 1A1-and 2C19-mediated drug oxidations were significantly and dose-dependently suppressed by IL-1β, under the present limited conditions. These results suggest that cytokines can influence hepatic P450 mRNA expression levels in cynomolgus macaques, just as cytokines are reported to affect P450 expression in humans., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Common marmoset (Callithrix jacchus) is an attractive animal model primate species for potential use in drug metabolism and pharmacokinetic studies. In this study, marmoset cytochrome P450 (P450) 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 cDNAs were isolated from marmoset tissues (brains, lungs, livers, kidneys, and jejunums). Deduced amino acid sequences (89-98% homologous) of the marmoset P450 gene suggested similarity of molecular characteristics of marmoset P450s to human counterparts, compared with those of pig, rabbit, and rodents. Phylogenetic analysis using amino acid sequences indicated 11 marmoset P450 forms clustered with those of human and other primate counterparts, suggesting marmoset P450s have an evolutionary close relationship to human and other primate counterparts. Tissue expression patterns of these P450 mRNAs except for P450 7B1 mRNA were generally similar to those of human P450s in the five tissue types analyzed. These results suggest similarity of molecular characteristics for P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 between marmosets and humans, in addition to the orthologs of human P450 1, 2, 3, and 4 families previously identified and characterized in marmosets., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The cynomolgus macaque is an important nonhuman primate species in drug metabolism studies, in part because of its evolutionary closeness to humans. Cytochromes P450 (P450s) have been investigated in the major drug-metabolizing organs, i.e., the liver and small intestine, but have not been fully investigated in the brain. However, recent investigations have indicated possible important roles for P450s in the brain. In this study, by using the quantitative polymerase chain reaction, we measured the mRNA levels of 38 cynomolgus drug-metabolizing enzymes, including 19 P450s, 10 UDP-glycosyltransferases, and 9 other enzymes, in 11 brain regions. Among these drug-metabolizing enzymes, expression of 32 enzyme mRNAs were detected in one or more brain regions, indicating their possible roles in the brain. Further investigation of metabolic activities would facilitate better understanding of the importance of these enzymes in the brain., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Polymorphic human flavin-containing monooxygenase (FMO) 3 is an important drug-metabolizing enzyme for nitrogen- or sulfur-containing compounds. Cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, have corresponding FMO3 molecular and enzymatic similarities to humans; however, genetic polymorphisms have not been investigated in macaques. In this study, re-sequencing of FMO3 in 64 cynomolgus and 32 rhesus macaques found a total of 18 non-synonymous variants. Nine variants were unique to cynomolgus macaques, of which 4 (including Q506K) were found only in Indochinese, 4 (including V299I, E348H, and G530A) only in Indonesian lineages, and one was common. Other five variants (including S504T at >10% allele frequencies) were unique to rhesus macaques. By functional characterization using cynomolgus FMO3 proteins heterologously expressed in Escherichia coli, FMO3 R509H variant appeared to suppress methimazole and benzydamine S- or N-oxygenations. Seven variants showed substantially lower benzydamine N-oxygenation as compared with wild-type FMO3 protein. Further analysis indicated that two of these variants, FMO3 G530A and R417H, showed significantly lower benzydamine N-oxygenation in liver microsomes of the homozygotes as compared with wild-type animals. Therefore, inter-animal variability of FMO3-dependent drug metabolism is at least partly accounted for by genetic polymorphisms in cynomolgus and rhesus macaques, similar to humans., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Animal species used in the preclinical studies for development of monoclonal antibody (mAb) drugs are surveyed in this review. Relevant animal species for preclinical studies of mAb candidates are those express desired epitope of mAb candidates. Cynomolgus monkeys cross-react with mAb drugs much higher than other animal species commonly used in preclinical studies such as absorption, distribution, metabolism and excretion (ADME), efficacy, and toxicity studies, for development of new drugs. Moreover, plasma exposure of the mAb drugs in humans is predicted well from the exposure in the monkeys, and the placental transfer of immunoglobulin G (IgG, all the mAb drugs contain IgG) from mother to fetus is similar between humans and the monkeys from a viewpoint of time course and plasma level of IgG transferred. These observed findings indicate that the monkeys are the most suitable animal species used in the ADME and toxicity studies for development of new mAb drugs., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cynomolgus macaques are an important primate species for drug metabolism studies; however cynomolgus CYP2C76, an important drug-metabolizing enzyme, accounts for drug metabolism differences to humans, so that CYP2C76-null animals might show drug-metabolizing properties more similar to humans. In this study, attempts were made to produce CYP2C76-null animals by assisted reproduction technology. Oocytes and sperm collected from the heterozygotes for the null allele (c.449TG > A) were subjected to intracytoplasmic sperm injection, and the embryos produced were cultured in vitro through the blastocyst stage. Preimplantation genetic diagnosis using a biopsied portion of the blastocyst revealed that none of the 32 blastocysts analyzed were homozygotes. In contrast, 2 of the 20 embryos analyzed were homozygotes at the 8-cell stage, indicating that CYP2C76-null embryos most likely stop developing between the 8-cell and blastocyst stage. By polymerase chain reaction, expression of CYP2C76 mRNA was detected in oocytes and blastocysts, but not in 2-, 4-, 8-, or 16/32-cell stage embryos. Metabolic assays showed that CYP2C76 metabolized progesterone. These results indicated that CYP2C76 null was likely embryonic lethal, suggesting its potential role during early embryogenesis in cynomolgus macaques., (Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Sex and age differences in hepatic expression of drug-metabolizing enzyme genes could cause variations in drug metabolism, but has not been fully elucidated, especially in Asian population. In this study, the global expression of human hepatic genes was analyzed by microarrays in 40 Japanese subjects (27 males and 13 females). Thirty-five sex-biased genes were identified (P < 0.005). Whereas, 60 age-biased genes in two age groups, <60 years and ≥70 years (P < 0.001), were identified in males. By Gene Ontology analysis, the sex-biased genes were related to protein catabolism and modification, while the age-biased genes were related to transcription regulation and cell death. Quantitative polymerase chain reaction confirmed the female-biased expression of drug-metabolizing enzyme genes BChE, CYP4X1, and SULT1E1 (≥1.5-fold, P < 0.05). Further analysis of drug-metabolizing enzyme genes indicated that expression of CYP2A6 and CYP3A4 in females in the ≥70 age group was less than in the <60 age group (≥1.5-fold, P < 0.05), and this trend was also observed for PXR expression in males (≥1.5-fold, P < 0.05). The results presented provide important insights into hepatic physiology and function, especially drug metabolism, with respect to sex and age., (Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×10 6 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

A collaborative study of the endogenous phosphatidylinositol glycan class A (Pig-a) gene mutation assay was conducted by the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group with a single-dosing regimen of test chemicals administered to male rats. As a part of the study, two DNA alkylating agents, methylnitrosourea (MNU) and temozolomide (TMZ), were dosed by single oral gavage at 25, 50, and 100mg/kg body weight. Pig-a mutant analysis of total red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay) was performed on Days 8, 15 and 29 after the administration. Both chemicals increased Pig-a mutants among RBCs and RETs with dose dependency on all days examined. The mutant frequencies were higher among RETs compared with RBCs, indicating that the PIGRET assay could detect mutagenicity more sensitively than the RBC Pig-a assay after a single dose of test chemicals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay were reported and analyzed statistically using the simple means of % tail DNA. However, OECD test guideline TG 489 recommends use of the median for data analysis due to the hierarchical nature of the data. Comparison between the simple mean approach and the median based approach for positive/negative/equivocal chemical calls was conducted using the % tail DNA data for the 40 chemicals tested in the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay, using liver and stomach as target organs. In the liver, two genotoxic chemicals, o-anisidine and 9-aminoacridine hydrochloride monohydrate, were positive using the median based approach but negative using the simple mean approach, and two genotoxic chemicals, 2-acetylaminofluorene and busulfan were equivocal using the median based approach but negative using the simple mean approach. In contrast, cadmium chloride (genotoxic carcinogen) was equivocal in both organs using the median based approach, while positive and equivocal in liver and stomach, respectively, using the simple mean approach. Two data sets of sodium arsenite showed equivocal and negative results for liver using the median based approach, although both data sets were equivocal using the simple mean approach. Overall, there are no large differences in terms of the genotoxic call between both approaches. However, the median based approach recommended in OECD TG 489 has an advantage toward higher precision within the groups treated with a test chemical, whereas the approach might show the lower values for the effect., (Copyright © 2015. Published by Elsevier B.V.)

The Japanese Center for the Validation of Alternative Methods (JaCVAM) sponsored an international prevalidation and validation study of the in vivo rat alkaline pH comet assay. The main objective of the study was to assess the sensitivity and specificity of the assay for correctly identifying genotoxic carcinogens, as compared with the traditional rat liver unscheduled DNA synthesis assay. Based on existing carcinogenicity and genotoxicity data and chemical class information, 90 chemicals were identified as primary candidates for use in the validation study. From these 90 chemicals, 46 secondary candidates and then 40 final chemicals were selected based on a sufficiency of carcinogenic and genotoxic data, differences in chemical class or genotoxic or carcinogenic mode of action (MOA), availability, price, and ease of handling. These 40 chemicals included 19 genotoxic carcinogens, 6 genotoxic non-carcinogens, 7 non-genotoxic carcinogens and 8 non-genotoxic non-carcinogens. "Genotoxicity" was defined as positive in the Ames mutagenicity test or in one of the standard in vivo genotoxicity tests (primarily the erythrocyte micronucleus assay). These chemicals covered various chemicals classes, MOAs, and genotoxicity profiles and were considered to be suitable for the purpose of the validation study. General principles of chemical selection for validation studies are discussed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

At the 6th International Workshop on Genotoxicity Testing (IWGT), the liver micronucleus test working group discussed practical aspects of the in vivo rodent liver micronucleus test (LMNT). The group members focused on the three methodologies currently used, i.e., a partial hepatectomy (PH) method, a juvenile/young rat (JR) method, and a repeated-dose (RD) method in adult rodents. Since the liver is the main organ that metabolizes chemicals, the LMNT is expected to detect clastogens, especially those that need metabolic activation in the liver, and aneugens. Based on current data the three methods seem to have a high sensitivity and specificity, but more data, especially on non-genotoxic but toxic substances, would be needed to fully evaluate the test performance. The three methods can be combined with the micronucleus test (MNT) using bone marrow (BM) and/or peripheral blood (PB). The ability of the PH method to detect both clastogens and aneugens has already been established, but the methodology is technically challenging. The JR method is relatively straightforward, but animal metabolism might not be fully comparable to adult animals, and data on aneugens are limited. These two methods also have the advantage of a short testing period. The RD method is also straightforward and can be integrated into repeated-dose (e.g. 2 or 4 weeks) toxicity studies, but again data on aneugens are limited. The working group concluded that the LMNT could be used as a second in vivo test when a relevant positive result in in vitro mammalian cell genotoxicity tests is noted (especially under the condition of metabolic activation), and a negative result is observed in the in vivo BM/PB-MNT. The group members discussed LMNT protocols and reached consensus about many aspects of test procedures. However, data gaps as mentioned above remain, and further data are needed to fully establish the LMNT protocol., (Copyright © 2014 Elsevier B.V. All rights reserved.)

At the 6th International Workshop on Genotoxicity Testing, the liver micronucleus test (MNT) working group briefly discussed the MNT using tissues other than liver/erythrocytes. Many tissues other than liver/erythrocytes have been studied, primarily for research purposes. They have included the colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. These tissues were chosen because they were target sites of carcinogens, and/or relevant to a specific route of exposure. Recently, there has been particular focus on the gastrointestinal (GI) tract as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. A collaborative study of the rat glandular stomach and colon MNT was conducted in conjunction with a collaborative study of the repeated-dose liver MNT. Based on limited data currently available, the rodent MNT using the glandular stomach and/or colon seems to detect genotoxic carcinogens with GI tract target-organ specificity. The working group concluded that the GI tract MNT would be a promising method to examine clastogenicity or aneugenicity of test chemicals in the stomach and/or colon. Further data will be needed to fully establish the methods, and to identify the sensitivity and specificity of the GI tract MNT., (Copyright © 2015 Elsevier B.V. All rights reserved.)

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use., (Copyright © 2014 Elsevier B.V. All rights reserved.)

MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that competes with the binding of the PARP substrate nicotinamide adenine dinucleotide (NAD), is being developed as a neuroprotective agent against acute ischemic stroke. MP-124 increased structural chromosomal aberration in CHL/IU cells, but showed negative results in the bacterial reverse mutation test, and the rat bone marrow micronucleus (MN) and the rat liver unscheduled DNA synthesis tests after the intravenous bolus injection. Thus, MP-124 did not appear to be direct-acting mutagen. Since, PARP-1 is a key enzyme in DNA repair, the effect of continuous PARP-1 inhibition by MP-124 was further examined in the rat MN test under 24-h intravenous infusion, and an increase in micronucleated immature erythrocytes (MNIE) was observed. The increase was clearly reduced by co-treatment with nicotinic acid, which resulted in increased intracellular NAD levels. This is consistent with the established activity of MP-124 as a competitive inhibitor of PARP and provides strong evidence that the DNA-damaging effect that leads to the increase in MNIE is a secondary effect of PARP-1 inhibition. This mechanism is expected to result in a threshold for the induction of MNIE by MP-124, and allows for the establishment of a safe margin of exposure for the therapeutic use of MP-124., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A repeated-dose liver micronucleus assay using young adult rats was conducted with methyl methanesulfonate (MMS) as a part of a collaborative study supported by the Collaborative Study Group for the Micronucleus Test/the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. MMS is a classical DNA-reactive carcinogen, but it is not a liver carcinogen. In the first experiment (14-day study), MMS was administered per os to 6-week-old male Crl:CD (SD) rats every day for 14 days at a dose of 12.5, 25, or 50mg/kg/day. In the second experiment (28-day study), 6-week-old male SD rats were treated with MMS at 7.5, 15, or 30mg/kg/day for 28 days, because the highest dose used in the 14-day study (50mg/kg/day) caused mortality. Hepatocyte and bone marrow cell specimens were prepared on the day after the final dose. The frequency of micronucleated hepatocytes (MNHEPs) in the liver and that of micronucleated immature erythrocytes (MNIMEs) in the bone marrow were evaluated. Exposure to 50mg/kg/day MMS for 14 days resulted in an increased frequency of MNHEPs, but MMS had no effect on the frequency of MNHEPs in the rats exposed to the chemical for 28 days at doses up to 30mg/kg/day. MMS induced MNIMEs production at doses of 25 and 50mg/kg/day in the 14-day study and at doses of 15 and 30mg/kg/day in the 28-day study. Overall, the effect of MMS on the frequency of MNHEPs was considered to be equivocal., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cynomolgus and rhesus macaques are non-human primate species widely used in drug metabolism studies. Cynomolgus CYP2C9 (formerly known as CYP2C43) is predominantly expressed in liver and encodes a drug-metabolizing enzyme that metabolizes human CYP2C substrates such as S-mephenytoin and progesterone. In addition, cynomolgus CYP2C9 also metabolizes caffeine, resulting in the formation of the metabolite that is not generated efficiently in humans. Genetic variants of human CYP2C genes account for the inter-individual variability in drug metabolism: however, CYP2C9 variants have not been found in macaques. To see if CYP2C9 is polymorphic in macaques, in this study, CYP2C9 was re-sequenced in 78 cynomolgus and 36 rhesus macaques. A total of 27 non-synonymous variants were found, among which 4 were located in substrate recognition sites, the domain important for protein function. Thirteen and seven variants were unique to cynomolgus and rhesus macaques, respectively. This study revealed the polymorphic nature of cynomolgus and rhesus CYP2C9, similar to human CYP2C genes, by identification of numerous genetic variants including non-synonymous variants., (Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Background: The relationship between atrioventricular valve regurgitation (AVVR) and valve annulus after bidirectional cavopulmonary anastomosis (BCPA) and adequate indications for valve repair are unclear., Methods: We evaluated the size of the valve annulus and the grade of AVVR before and immediately after BCPA, and at the most recent follow-up before the Fontan operation in 37 patients with a functional single ventricle., Results: Nine patients underwent concomitant valve surgery. The mean z value of the valve annulus was significantly lower postoperatively than preoperatively in the 28 patients who were not treated by valve surgery (0.45 vs 1.51, p=0.01). However, mean regurgitation scores did not significantly change after BCPA (1.60 vs 1.78, p=0.08). The most recent assessment showed that the mean z value increased compared with that immediately after BCPA (1.36 vs 0.45, p=0.005). This increase was significant in the patients with moderate regurgitation. The mean z value of the valve annulus of the patients treated by concomitant valvuloplasty was significantly lower postoperatively than preoperatively (-0.25 vs 3.9, p=0.0001) and remained low at the latest evaluation. Mean regurgitation scores also significantly decreased after BCPA (2.25 vs 3.37, p=0.007)., Conclusions: Unloading the systemic ventricle by BCPA leads to a decrease in the relative size of the atrioventricular valve. However, this decrease does not improve the degree of AVVR in the absence of concomitant valve repair. Concomitant valve repair is justified in patients with moderate or worse AVVR and an abnormal valve structure., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Neonatal Fc receptor (FcRn), a heterodimer of MHC class I-like protein and β2-microglobulin, encoded by FCGRT and B2M, respectively, is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of IgG. Cynomolgus macaques are important animal species used in the evaluation of therapeutic antibodies, largely due to sequence similarities of target proteins to those of humans. Because the function of FcRn could be modified by mutations in FCGRT or B2M, 71 cynomolgus and 24 rhesus macaques were analyzed in the present study. A total of 21 variants were identified, of which 4 were non-synonymous in FCGRT. Fifteen variants were unique to cynomolgus macaques, of which 3, 2, and 5 were unique to cynomolgus macaques bred in China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN), respectively. Five variants were shared by MacfaCHN and MacfaCAM, but not by MacfaIDN. In B2M, only 5 variants were found, including 2 non-synonymous variants. Tissue expression analysis showed that cynomolgus FCGRT and B2M were widely expressed in the 10 tissue types analyzed. None of the non-synonymous variants of FCGRT or B2M found changes in the amino acid residues known to be important for FcRn function, suggesting that substantial inter-animal variability of FcRn is not expected for the cynomolgus macaques analyzed.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (<2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (<2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.

Japanese quail (Coturnix japonica) are farmed worldwide as poultry. Quail have been used as experimental animals in various scientific fields, but their immunological characteristics have not been well characterized. In this study, to develop a method for analyzing the innate immune response of quail to infectious pathogens, we determined the nucleotide sequences of major interleukins (IL) and Toll-like receptor (TLR)-7 of quail and developed quantitative real-time PCR assays. The nucleotide sequences of quail IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12a, IL-12b, IL-13, IL-18, and TLR-7 were determined based on the sequences of the chicken genes. Specific primers for each of these genes and previously reported interferon (IFN)-α, IFN-γ, and IL-2 genes were designed for quantitative real-time PCR. Standard curves for quantification were established using serial dilutions of external standard plasmids containing real-time PCR products. Then, real-time PCR was performed to monitor the kinetics of quail immune-related gene expression induced in splenocytes stimulated with concanavalin A. After amplification, the r(2) values of the standard curves for all target genes were above 0.980. Melting analysis of real-time PCR revealed specific amplification of each gene that could be visualized clearly as a single peak of melting temperature in a melt peak chart. These data show that the mRNA expressions of quail immune-related genes can be accurately quantified using this real-time PCR assay. In this study, we showed the nucleotide sequences of several quail cytokine mRNA and constructed the quantitative real-time PCR for quail immune-related genes.

Cynomolgus CYP2C76, not orthologous to any human cytochrome P450, partly accounts for species differences in drug metabolism between cynomolgus macaques and humans. To discover the CYP2C76 variants, we previously surveyed cynomolgus macaque genomes and found several non-synonymous variants, including a null allele. However, the analysis was limited to cynomolgus macaques, and the number of genomes was relatively small. In this study, therefore, further screening was conducted using 74 cynomolgus and 30 rhesus macaques. A total of 18 non-synonymous variants was found, among which 7 were in substrate recognition sites, important for protein function, and 14 (74%) were shared by both macaque lineages. In cynomolgus macaques, 3 (16%) non-synonymous variants were unique to Indochinese animals, whereas all the variants found in Indonesian animals were shared by Indochinese animals. Among the 18 variants, as compared with the wild type, in progesterone 16α-hydroxylation, L65F, M310L, and N364S variants showed lower metabolic activity and lower intrinsic clearance by kinetic analysis. Molecular modeling indicated that the reduced catalytic activity of the L65F variant in progesterone 16α-hydroxylation possibly resulted from a longer distance of progesterone to the heme in the active site of the CYP2C76 protein. L65F, M310L, and N364S variants might partly influence inter-animal variations of CYP2C76 metabolic activities.