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Study on chromosome analyses of human giant oocytes by micromanipulation and next-generation sequencing
Get PDF麻布大学博士(学術)【序論】ヒト生殖補助医療（assisted reproductive technology: ART）は、不妊症に対する治療、癌治療後の妊孕性保全などの目的で、医療介在によって生殖活動を補助するものである。ヒトARTでは卵巣からの卵採取が重要であるが、ごく稀に採卵時に通常の卵母細胞よりも直径が大きな「giant oocytes : GOs」が採取されることがある。このGOsの多くは2倍体で、受精させても染色体の数的異常胚を形成する。GOsが採卵される確率は0.12から0.3％であり、卵巣刺激法や患者の体質に依存しないと考えられている。GOsは、通常の卵母細胞と同じ受精能を示す。また、GOsはMⅡ期に達した際、極体（polar body: PB）2個およびchromosome-spindle complex (CSC) 2個を有するものと、PB1個およびCSC1個を有するものが存在する。GOsは受精後のその初期胚の染色体構成に異数性が生じることが報告されているが、その詳細については不明な点が多い。成熟卵母細胞のPBは減数分裂の産物で、染色体構成はCSCと相同であることから、GOsのPBの染色体解析ができれば、対応するGOの細胞質内の染色体も間接的に解析でき、受精およびその後の初期胚発生に有用な情報となりうる。染色体解析法については、染色体標本を作製し、特殊な染色を施す従来法でない次世代シーケンス (next-generation sequencing: NGS) 法による解析が最近開発され、胚の着床前診断にも適用されている。
本研究はヒトGOsにおける染色体解析を目的に、GOsのPBおよびCSCを個々にサンプリングするためのマイクロマニピュレーション法を開発し、ヒトGOsへ適用して採取ゲノムサンプルのNGS法による染色体解析を試みた。
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第一章 マウスMⅡ期卵母細胞におけるpolar body (PB)およびmetaphase Ⅱ chromosome-spindle complex (CSC) のマイクロマニピュレーションおよび次世代シーケンス法を用いた染色体解析
【目的】卵母細胞におけるPBゲノムおよびCSCゲノムの正確なサンプリング技術は、本研究に非常に重要である。同一卵母細胞質内にCSCが2つ存在し、かつそれぞれのCSCsの近傍に対応するPBsを有するGOの染色体解析には、マイクロマニピュレーションを用いたPBおよびCSCのサンプリングが適していると考えられる。本章では、マウス卵母細胞を用いてPBおよびCSCを採取し、NGS法による染色体数の解析を試みた。
【方法】超低温保存マウス未受精卵を購入し、その加温卵のPBおよびCSCを、倒立顕微鏡下のマイクロマニピュレーションにより採取して、染色体解析に適用しうる状態で採取できるか検討した。すなわち、PBの近傍の透明帯をレーザーにて穿孔し、PBをガラスピペットで吸引採取してサンプルチューブへ移して凍結保存した。続けてCSCを倒立顕微鏡の偏光装置にて確認しながらガラスピペットで吸引採取し、凍結保存した。採取したPBsおよびCSCsを含んだサンプルチューブを株式会社アイジェノミクス・ジャパン（東京）へ送付し、NGS法による染色体解析を依頼した。
【結果】マウス成熟卵母細胞20個に対し、全ての卵母細胞からPBsおよびCSCsの採取ができた。NGS法による染色体解析成功率は、CSCs由来染色体、PBs由来染色体それぞれ100% (5/5)、60% (3/5) であった 。本手法で採取したマウス卵母細胞のCSCsおよびPBsの染色体解析はNGS法により解析可能であった。
\n第二章 ヒト生殖補助医療におけるGOsの採卵率
【目的】GOsはヒトを含めた哺乳類に存在することが報告されているが、ヒトART において採取されたGOsについて、それらの核の状態や成熟能などの詳細な解析はされていない。そこで、ヒトARTにおけるGOs採卵成績および核の状態などについて、山下湘南夢クリニックの採卵成績から調べた。
【方法】本章は山下湘南夢クリニック倫理審査委員会および国立国際医療研究センター倫理委員会にて承認を得て実施した。山下湘南夢クリニックの自然周期または低卵巣刺激周期の採卵成績を対象としてGOs採取率を調べた。得られた卵母細胞は倒立顕微鏡下にて観察し、核の状態（卵母細胞の成熟）および卵母細胞の細胞質の最大直径を測定し、その直径が140 µmを超えるものをGOと判定した。採取されたGOsは、文書同意が得られたものを本章の対象症例とした。
【結果】採卵を行った6124周期（クロミフェン周期：3170、レトロゾール周期：1429、自然周期：1525） において、10392個の卵母細胞が得られ、GOsの採卵率は0.29% (30 GOs /10392 oocytes) であった。卵巣刺激法ごとのGO採卵率は、クロミフェン周期、レトロゾール周期、自然周期においてそれぞれ0.30% (20 GOs/6606 oocytes)、0.29% (7 GOs/2401 oocytes) 、0.21% (3 GOs/1405 oocytes) であり、卵巣刺激法による差は認められなかった(P > 0.05)。得られたGOsはMⅡ期が53.3%(16/30)、MI期が20.0% (6/30)、GV期が26.7% (8/30)であった。
\n第三章 ヒトGOsの体外成熟培養
【目的】未成熟の状態（GV期もしくはMI期）で得られたGOsは体外成熟培養を行い、その体外成熟能について調べた。
【方法】採卵されたMI期およびGV期GOsについて、採卵後37.5℃、6% CO2、5% O2、89% N2の気相でガス平衡させたoocyte maturation-medium (IVM Media KIT, Cooper Surgical, Inc. USA) を用いて35 mm dish (AGC TECHNO GLASS, Japan) に20 µL dropを作製し、OVOIL (Vitrolife, Sweden) で覆い、インキュベーター（ES6S, 株式会社アステック, 福岡）内で冠状細胞が付いた状態で24時間成熟培養を行った。成熟培養後、GOsは冠状細胞を除去後、PBsおよびCSCsの数を倒立顕微鏡の偏光装置を用いて確認した。成熟培養成績については、2015年10月1日から2017年12月31日の期間に山下湘南夢クリニックで実施された採卵において、同条件で成熟培養した通常の卵母細胞と比較した。
【結果】MI期で得られた6個のGOsは成熟培養により全てが成熟し、通常卵の成熟率89.4% (2079/2326) と差は認められなかった(P > 0.05)。成熟したGOsのうちPBを2つ有するGOsが83.3% (5/6)、そのうちCSCを2つ有するものは80.0% (4/5)であった。GV期で得られた8個のGOsのうち、7個について成熟培養を行い、71.4% (5/7) が成熟し、通常卵の成熟率65.1% (829/1274)と差は認められなかった(P > 0.05)。成熟した5個のGOsは全てPBを2つ有しており、そのうちCSCを2つ有するものは4個であった。本検討の結果、未成熟で得られたGOsの成熟率は、通常卵と比較して差が認められないこと(P > 0.05)、成熟したGOsは80%以上の割合でPBを2つ有することが示された。
\n第四章 ヒトGOsのNGS法による染色体解析
【目的】ヒトGOsからマイクロマニピュレーションでPBsおよびCSCsを採取し、NGS法による染色体解析を試みた。
【方法】採卵時に成熟しておりPBおよびCSCを2つずつ有するGOsをガラス化保存・加温し、本検討に使用した。偏光顕微鏡にてCSCの位置をそれぞれ確認し、片方のPB近傍に穿孔し、ガラスピペットでPBを吸引採取し凍結保存した。続けて、採取したPBの近傍に位置するCSCをピペットで吸引採取し、サンプルチューブへ移して凍結保存した。1組のPB、CSCを採取した後、残りのPB、CSCについても、同様の手順で採取し、凍結保存を行った。2つのPB、CSCを採取した後、透明帯を完全に除去してcytoplastをサンプルチューブに移して凍結保存した。サンプルチューブは株式会社アイジェノミクス・ジャパン（東京）へ送付し、NGS法による染色体解析を依頼した。
【結果】GOs 4個の、合計8個のPBsについて全ての採取に成功 (8/8, 100%) した。また、合計8個のCSCsについては7個の採取に成功 (7/8, 87.5%) した。3個のGOsのPBsおよびCSCsをNGS法により解析した結果、全ての採取サンプルに染色体異数性が確認された。本検討により、PBおよびCSCをマイクロマニピュレーションにて直接採取し、NGS法により染色体解析ができることが示された。
本研究で確立したマイクロマニピュレーションおよびNGS法を適用してヒトGOsのPBsおよびCSCsの染色体解析ができることが示された。また、CSCと近接するPBの染色体数的異常は相補的であった。従ってヒトGOsについては、胚移植に用いる配偶子の対象からは除外するべきである。doctoral thesi
Highly successful production of viable mice derived from vitrified germinal vesicle oocytes
Get PDFThe vitrification of immature germinal vesicle (GV) oocytes is an important way to preserve genetic resources and female fertility. However, it is well known that cryopreserved GV oocytes have very poor developmental ability and that further improvement in this technique is needed. We previously reported the successful vitrification of matured mouse oocytes with enclosed cumulus cells using the calcium-free vitrification solution supplemented with ethylene glycol (EG) by the minimal volume cooling (MVC) method. In this study, we investigated whether our method is applicable to the vitrification of mouse oocytes at the GV stage (GV oocytes). Following maturation and fertilization in vitro, vitrified GV oocytes showed high survival (94.3 ± 2.0%) and maturation (94.3 ± 2.1%) rates. Although the fertilization and blastocyst rates of vitrified oocytes (fertilization: 46.6 ± 4.9% and blastocyst: 46.6 ± 3.0%) were significantly lower than those of fresh oocytes (fertilization: 73.0 ± 7.1% and blastocyst: 71.6 ± 8.0%) (P 0.05). In conclusion, we here show, for the first time, the efficient production of live mice derived from vitrified GV oocytes.journal articl
Usefulness of the MALDI-TOF MS technology with membrane filter protocol for the rapid identification of microorganisms in perioperative drainage fluids of hepatobiliary pancreatic surgery
Get PDFSurgical site infections (SSIs) are significant and frequent perioperative complications, occurring due to the contamination of the surgical site. The late detection of SSIs, especially organ/space SSIs which are the more difficult to treat, often leads to severe complications. An effective method that can identify bacteria with a high accuracy, leading to the early detection of organ/space SSIs, is needed. Ninety-eight drainage fluid samples obtained from 22 patients with hepatobiliary pancreatic disease were analyzed to identify microorganisms using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) with a new membrane filtration protocol and rapid BACpro® pretreatment compared to sole rapid BACpro® pretreatment. The levels of detail of rapid BACpro® pretreatment with or without filtration were also evaluated for the accuracy of bacterial identification. We found that reliable scores for E. coli and E. faecalis were obtained by inoculation with 1.0 × 104 CFU/ml after preparation of the membrane filter with rapid BACpro®, indicating approximately 10-folds more sensitive compared to sole rapid BACpro® pretreatment in drainage fluid specimens. Among 60 bacterial positive colonies in drainage fluid specimens, the MALDI-TOF MS and the membrane filtration with rapid BACpro® identified 53 isolates (88.3%) with a significantly higher accuracy, compared to 25 isolates in the rapid BACpro® pretreatment group (41.7%) (p < 0.001). Among the 78 strains, 14 enteric Gram-negative bacteria (93.0%) and 55 Gram-positive cocci (87.3%) were correctly identified by the membrane filtration with rapid BACpro® with a high reliability. This novel protocol could identify bacterial species within 30 min, at 3 per sample, thus leading to cost and time savings. MALDI-TOF MS with membrane filter and rapid BACpro® is a quick and reliable method for bacterial identification in drainage fluids. The shortened analysis time will enable earlier selection of suitable antibiotics for treatment of organ/space SSIs to improve patients’ outcomes.journal articl
Timing urinary tract reconstruction in rats to avoid hydronephrosis and fibrosis in the transplanted fetal metanephros as assessed using imaging
Get PDFChronic kidney disease leads to high morbidity rates among humans. Kidney transplantation is often necessary for severe symptoms; however, options for new curative treatments are desired because of donor shortage. For example, it has been established that the kidneys can efficiently generate urine after transplantation of the metanephros, ureter, and bladder as a group. After transplantation, the urine can indirectly flow into the recipient’s bladder using a stepwise peristaltic ureter system method where the anastomosis is created via the recipient’s ureter for urinary tract reconstruction. However, the growth of the regenerated metanephros varies significantly, whereas the time window for successful completion of the stepwise peristaltic ureter system that does not cause hydronephrosis of the metanephros with bladder (ureter) is quite narrow. Therefore, this study was conducted to periodically and noninvasively evaluate the growth of the transplanted metanephros, ureter, and bladder in rats through computed tomography and ultrasonography. The ultrasonographic findings highly correlated to the computed tomography findings and clearly showed the metanephros and bladder. We found that the degree of growth of the metanephros and the bladder after transplantation differed in each case. Most of the rats were ready for urinary tract reconstruction within 21 days after transplantation. Optimizing the urinary tract reconstruction using ultrasonography allowed for interventions to reduce long-term tubular dilation of the metanephros due to inhibited overdilation of the fetal bladder, thereby decreasing the fibrosis caused possibly by transforming growth factor-β1. These results may be significantly related to the long-term maturation of the fetal metanephros and can provide new insights into the physiology of transplant regeneration of the metanephros in higher animals. Thus, this study contributes to the evidence base for the possibility of kidney regeneration in human clinical trials.journal articl
Malaria in the postpartum period causes damage to the mammary gland
Get PDFMastitis is an inflammation of the mammary gland in the breast and is typically due to bacterial infection. In malaria-endemic areas, mastitis with accompanying fever can be challenging to differentiate from malaria. At the same time, it is unclear whether malaria infection is directly involved in the development of mastitis. In the present study, whether mastitis develops during infection with malaria parasites was investigated using a rodent malaria model with Plasmodium berghei (P. berghei; Pb) ANKA. The course of parasitemia in postpartum mice infected with Pb ANKA was similar to the course in infected virgin mice. However, infected postpartum mice died earlier than did infected virgin mice. In addition, the weight of pups from mice infected with Pb ANKA was significantly reduced compared with pups from uninfected mice. The macroscopic and histological analyses showed apparent changes, such as destruction of the alveolus wall and extensive presence of leukocytes, in mammary gland tissue in mice infected during the postpartum period. The findings suggest that women during the postpartum period are more vulnerable to complications when infected with malaria parasites, particularly women who do not acquire protective immunity against malaria parasites. Based on the proteomic analysis, IFN-γ signaling pathway-related proteins in mammary gland tissue of the infected postpartum mice were increased. Our results indicate that inflammation induced by IFN-γ, a proinflammatory cytokine, may contribute to negative histological changes in mammary gland tissue of postpartum mice infected with Pb ANKA. In IFN-γ receptor 1-deficient (IFNGR1-KO) mice, the histological changes in mammary gland tissue of the infected postpartum wild-type mice were improved to almost normal mammary gland structure. Furthermore, weight loss in pups delivered by infected IFNGR1-KO postpartum mice was not observed. Taken together, these findings indicate that inflammation induced by IFN-γ is associated with development of mastitis in postpartum mice infected with Pb ANKA. The present study results may increase our understanding of how disease aggravation occurs during postpartum malaria.journal articl
Toxicology study about metabolic interaction of rubber antioxidant 4-methyle or 5-methyle-2- mercaptobenzimidazole using in vitro assays
Get PDF麻布大学博士(獣医学)1. Introduction
The toxicity of chemical substances is greatly affected by their metabolism in the body. That is, chemical substances undergo detoxification, which weakens their toxicity, or activation, which increases their toxicity, when they are metabolized. On the other hand, cytochrome P450 (CYP), the enzyme that is quantitatively most abundant in the liver and is involved in the metabolism of many kinds of chemicals due to its low substrate specificity, can be induced or inhibited by chemicals. If the induced or inhibited CYPs are involved in the metabolism of other chemicals, a metabolic drug-drug interaction may occur in which the toxicity of the chemicals are affected. Therefore, toxicological evaluation of chemical substances should consider the effects of inducing or inhibiting CYPs and metabolic drug-drug interactions. However, currently there is no uniform method for evaluating metabolic drug-drug interactions, it is necessary to study each chemical substance individually.
Methylated-2-mercaptobenzimidazole in this study refers to 4-methyl-2-mercaptobenzimidazole (4-methyle-2-mercaptobenzimidazole, 4-MeMBI) and 5-methyl-2-mercaptobenzimidazole (5-methyle-2-mercaptobenzimidazole, 5-MeMBI), which are methylated derivatives of 2-mercaptobenzimidazole (2-mercaptobenzimidazole, MBI) at position 4 or 5. The industrially used 1:1 mixture of 4-MeMBI and 5-MeMBI is abbreviated as 4(5)-MeMBI, and when these methylated MBIs are collectively referred to as Methyl-MBIs.
4(5)-MeMBI and MBI are widely used industrially as anti-aging agents for rubber and are known to be environmental pollutants with low biodegradability. Therefore, various toxicological studies have been carried out due to concerns about their effects on human health.
By using rats, Methyl-MBIs and MBIs have been shown to have strong thyroid toxicity due to their thiourea structure. In repeated and single-dose oral administration studies in rats, the toxicity of 4(5)-MeMBI was weaker than that of MBI, and the peroxidase inhibitory activity, which indicates thyroid toxicity, was also weaker for 4- and 5-MeMBI than for MBI. 4-MeMBI was the weakest in these. Toxicity test using thyroid weight as an indicator showed weaker toxicity with the mixture 4(5)-MeMBI than with 5-MeMBI alone, suggesting that the toxicity may have been attenuated by the interaction of the test compounds in the mixture. On the other hand, no difference was observed between 4-MeMBI or 5-MeMBI and 4(5)-MeMBI in hepatotoxicity as an indicator of hepatomegaly, which was thought to be related to their effects in the liver. In a report on the effects of repeated administration on the activity and protein levels of hepatic CYPs, 4-MeMBI and 5-MeMBI strongly induced CYP1A and CYP2B, respectively, but 4(5)-MeMBI had no additive effects on 4-MeMBI and 5-MeMBI. Although these reports suggest that the relatively low toxicity of 4(5)-MeMBI is due to metabolic drug-drug interactions between 4-MeMBI and 5-MeMBI via CYP induction or inhibition, there is currently no uniform method of evaluation, so individual chemicals need to be studied. In this study, we aimed to clarify the metabolic drug-drug interactions induced by 4-MeMBI and 5-MeMBI. We investigated the cytotoxicity of Methyl-MBIs and MBIs using the rabbit cornea-derived cell line SIRC cells. Then, metabolism by CYPs and induction of CYPs were examined for 4-MeMBI and 5-MeMBI by in vitro assays using rat liver microsomes and primary cultured rat and human hepatocytes. These in vitro assays are expected to provide results that are easier to analyze and evaluate than in vivo tests.
2. Cytotoxicity of 4-methyl- and 5-methyl-2-mercaptobenzimidazole in SIRC cells
The cytotoxicity of MBI and Methyl-MBIs in SIRC were detected by the crystal violet staining assay. The half lethal concentration (LC50) was calculated from cell viability, and the intensity of cytotoxicity was found to be 5-MeMBI ≥ 4-MeMBI ≥ 4(5)-MeMBI > MBI. Methyl-MBIs showed similar cytotoxicity and were more potent than MBIs, suggesting that the presence of a methyl group on the benzene ring increases the cytotoxicity of MBI derivatives. In addition, SIRC cells have low drug-metabolizing enzyme activity, supporting the hypothesis that the low thyroid toxicity and hepatotoxicity of Methyl-MBIs in vivo are due to the induction of drug-metabolizing enzymes.
3. Metabolism of 4-MeMBI, 5-MeMBI and MBI in rat liver
The metabolism of 4-MeMBI, 5-MeMBI, and MBI by CYP in rat liver microsomes was investigated by HPLC method. The results showed that 4-MeMBI, 5-MeMBI, and MBI were metabolized by CYPs in a time-dependent manner. From the metabolic assay using liver microsomes treated with β-naphthoflavone, a CYP1A inducer, phenobarbital, a CYP2B inducer, and isoniazid, a CYP2E inducer, showed that 4-MeMBI and 5-MeMBI were metabolized by CYPs-dependent manner. CYPs 1A and 2B were involved in the metabolism of 4-MBI and 5-MeMBI, and both were more dependent on CYP2B. These results and the results of reported in vivo experiments suggest a metabolic drug-drug interaction in the metabolism of 4(5)-MeMBI, in which 5-MeMBI and 4-MeMBI are efficiently metabolized by CYP1A induced by 4-MeMBI and CYP2B induced by 5-MeMBI, respectively.
4. Effect of 4-MeMBI, 5-MeMBI and MBI on CYP activity in rats and human primary cultured hepatocytes
To further investigate the metabolic drug-drug interactions in 4(5)-MeMBI by induction of CYPs, we evaluated the induction of CYPs in primary cultured rat and human hepatocytes. As selected CYPs of CYP3A2/4 induction has not been confirmed in vivo tests but is known to be frequently involved in metabolic drug-drug interactions, and that of CYP1A1/2, which were strongly induced by 4-MeMBI in in vivo tests. Rat or human hepatocytes were cultured for 48 or 96 hours in the presence of 4-MeMBI, 5-MeMBI, or MBI. And then, the activity of CYP3A2/4 activity was determined by measuring 6β-hydroxylation of testosterone and that of CYP1A by CYP1A1/2 activity by measuring O-deethylation of 7-ethoxyresorufin using the HPLC method.
In rat hepatocytes, 4-MeMBI and 5-MeMBI inhibited CYP3A2, while 4-MeMBI strongly induced CYP1A1/2. MBI strongly inhibited CYP3A2 and weakly inhibited CYP1A1/2. These results were consistent with the reported in vivo tests results, suggesting that the induction by 4-MeMBI of CYP1A1/2, which metabolizes 4-MeMBI and 5-MeMBI, is important for the metabolic drug-drug interaction regarding the toxicity of 4(5)-MeMBI.
In human hepatocytes, weak induction of CYP1A and CYP3A4 by 4-MeMBI and 5-MeMBI was observed in a donor-dependent manner. These results suggest that metabolic drug-drug interactions related to the toxicity of 4(5)-MeMBI are unlikely to occur in humans.
5. Induction of CYPs mRNA by 4-MeMBI and 5-MeMBI in rat hepatocytes (additional data)
To investigate the effect of CYPs induction at the mRNA level, the rate of change of CYPs mRNA in rat’s oral administration with 4-MeMBI and 5-MeMBI for 8 consecutive days was evaluated using the Competitive RT-PCR. The results showed that 4-MeMBI and 5-MeMBI induced the mRNA expression of CYPs 1A2, 2B2, 2C11, 2E1, and 3A2. The induction of CYP1A2 was particularly large, and the amount of induction by 4-MeMBI was eight times higher than that by 5-MeMBI. Conversely, the induction of CYP2B2 by 5-MeMBI was 2-fold higher than that by 4-MeMBI. These results suggest that the increased activity of CYPs in in vivo and in vitro experiments is due to the induction of expression at the mRNA level.
6. Conclusion
In this study, we investigated the cytotoxicity of 4-MeMBI and 5-MeMBI, their metabolism by CYPs, and their induction of CYPs in liver using in vitro assays. The results showed that the strength of cytotoxicity of 4-MeMBI, 5-MeMBI and MBI was Methyl-MBIs > MBI which is opposite to the toxicity reported in vivo. 4-MeMBI and 5-MeMBI were shown to be metabolized by CYPs 1A and 2B and to induce CYP1A activity. Furthermore, we showed that the induction of CYPs activity by 4-MeMBI and 5-MeMBI was the mRNA level. These results suggest that 4-MeMBI and 5-MeMBI, when used as a mixture 4(5)-MeMBI, cause a metabolic drug-drug interaction that weakens thyroid toxicity and hepatotoxicity by promoting each other's metabolism through induction of CYPs. This is a useful phenomenon in that when highly toxic chemicals are used as a mixture, the toxicity is weakened by the metabolic drug-drug interaction of these chemicals.1. はじめに
化学物質による毒性は、その生体内における代謝により大きく影響される。すなわち、化学物質は代謝されることにより、毒性が弱まる解毒、または毒性が強まる活性化を受ける。一方、肝臓に量的に最も多く存在し、基質特異性が低いために、多種類の化学物質の代謝に関与する酵素であるチトクロームP450（CYP）は、化学物質によりその活性が誘導または阻害されることがある。誘導または阻害されたCYPが他の化学物質の代謝に関与する場合には、その化学物質の毒性が影響を受ける代謝的薬物間相互作用が起こる可能性がある。したがって、化学物質の毒性評価においては、CYPを誘導または阻害する作用および代謝的薬物間相互作用を考慮した毒性学的評価が必要である。しかしながら、代謝的薬物間相互作用については、現在のところ画一的な評価方法は存在しないため、個々の化学物質について研究的に調べる必要がある。
本研究におけるメチル化-2-メルカプトベンズイミダゾールとは、2-メルカプトベンズイミダゾール（2-mercaptobenzimidazole、MBI）の4位または5位のメチル化誘導体である4-メチル-2-メルカプトベンズイミダゾール（4-methyle-2-mercaptobenzimidazole、4-MeMBI）および5-メチル-2-メルカプトベンズイミダゾール（5-methyle-2-mercaptobenzimidazole、5-MeMBI）である。工業的に用いられる4-MeMBIおよび5-MeMBIの1対1混合物を4(5)-MeMBIと略称し、これらのメチル化MBIを総称する場合はMethyl-MBIsと略称する。
4(5)-MeMBIおよびMBIはゴム老化防止剤などとして工業的に幅広く利用されており、生分解性の低い環境汚染物質として知られている。そのため、ヒトの健康への影響が懸念されることから、様々な毒性学的研究が行われている。
Methyl-MBIsおよびMBIは、そのチオウレア構造による強い甲状腺毒性を有することが、ラットを用いて明らかにされている。ラットを用いた反復および単回投与試験では4(5)-MeMBIの毒性はMBIよりも弱く、甲状腺毒性を示すペルオキシダーゼ阻害活性もまた、4-および5-MeMBIはMBIよりも弱かった。4-MeMBIは最も弱かった。甲状腺重量を指標とした毒性評価では5-MeMBI単独よりも混合物4(5)-MeMBIで弱い毒性を示し、混合物内の被験物質における相互作用により毒性が減弱した可能性が示唆された。一方、肝肥大を指標とした肝毒性については4-MeMBIまたは5-MeMBIと4(5)-MeMBIとの間に差は認められず、肝臓における作用が関与するものと考えられた。反復投与による肝CYPの活性およびタンパク量への影響を調べた報告では、4-MeMBIはCYP1Aを、5-MeMBIはCYP2Bをそれぞれ強く誘導したが、4(5)-MeMBIでは4-MeMBIと5-MeMBIとによる相加作用は認められなかった。これらの報告から、4(5)-MeMBIの相対的に低い毒性は、4-MeMBIと5-MeMBIとのCYP誘導または阻害を介した代謝的薬物間相互作用によると考えられているものの、現在のところ画一的な評価方法は存在しないため、個々の化学物質について研究的に調べる必要がある。
本研究では、4-MeMBIおよび5-MeMBIによる代謝的薬物間相互作用を明らかにすることを目的とした。ウサギ角膜由来細胞株SIRC細胞を用いてMethyl-MBIsおよびMBIの細胞毒性を調べた。続いて、ラット肝ミクロゾーム、ラットおよびヒト初代培養肝細胞を用いたin vitro実験系により、4-MeMBIおよび5-MeMBIについて、CYPによる代謝、CYPの誘導を調べた。これらのin vitro実験系では、in vivo実験に比べて、解析および評価が容易な結果が得られることが期待される。
2．ウサギ角膜由来細胞株SIRC細胞におけるMBI、4-MeMBI、5-MeMBIおよび4(5)-MeMBIの細胞毒性
SIRC細胞におけるMBI、4-MeMBI、5-MeMBIおよび4(5)-MeMBIの細胞毒性をクリスタル・バイオレット染色性試験により検討した。細胞生存率から半数致死濃度（LC50）を算出した結果、細胞毒性の強度は5-MeMBI ≧ 4-MeMBI ≧ 4(5)-MeMBI ＞ MBIであることを明らかにした。Methyl-MBIsは類似した細胞毒性を示し、MBIよりも強い細胞毒性を示したことから、ベンゼン環にメチル基が存在するとMBI誘導体の細胞毒性が高まることを示唆している。さらに、SIRC細胞は薬物代謝酵素活性が低いことから、in vivoにおけるMethyl-MBIsの低い甲状腺毒性および肝毒性は薬物代謝酵素の誘導によるものであるという仮説を支持した。（Kuroda Y*, Miyajima A*, Sakemi-Hoshikawa K, Usami M, Mitsunaga K, Ohno Y, Sunouchi M. In vitro cytotoxicity of the thyrotoxic and hepatotoxic rubber antioxidant 2-mercaptobenzimidazole and its 4- or 5-methyl derivatives in rabbit corneal cells. Fundamental Toxicological Sciences. 2020, * These authors equally contributed to this work.）
3．ラット肝における4-MeMBI、5-MeMBIおよびMBIの代謝
ラット肝ミクロゾームにおける4-MeMBI、5-MeMBIおよびMBIについて、CYPによる代謝量をHPLC法により調べた。その結果、4-MeMBI、5-MeMBIおよびMBIは、時間依存的にCYPにより代謝されることが明らかになった。CYP1A誘導剤β-ナフトフラボン、CYP2B 誘導剤フェノバルビタールおよびCYP2E誘導剤イソニアジド処理した肝ミクロゾームを用いた実験から、4-MeMBIおよび5-MeMBIの代謝には、CYPs 1Aおよび2Bが関与し、いずれもCYP2Bにより依存的であることが明らかになった。以上の結果および報告されているin vivo実験の結果から、4(5)-MeMBIの代謝においては、4-MeMBIにより誘導されたCYP1Aおよび5-MeMBIにより誘導されたCYP2Bにより、5-MeMBIおよび4-MeMBIのそれぞれが効率的に代謝される、代謝的薬物間相互作用が示唆された。（Miyajima A*, Kuroda Y*, Sakemi-Hoshikawa K, Usami M, Mitsunaga K, Irie T, Ohno Y, Sunouchi M. In vitro metabolism of 4-methyl- and 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, in rat liver microsomes. Fundamental Toxicological Sciences. 2018, * These authors equally contributed to this work.）
4．ラットおよびヒト初代培養肝細胞における4-MeMBI、5-MeMBIおよびMBIのCYP活性に及ぼす影響
CYPの誘導による4(5)-MeMBIにおける代謝的薬物間相互作用をさらに調べるために、ラット初代培養肝細胞およびヒト初代培養肝細胞を用いてCYPの誘導を調べた。CYPとしてin vivo実験で誘導が確認されていないが肝における発現量が多く代謝的薬物間相互作用にしばしば関与することが知られているCYP3A2/4、およびin vivo実験で4-MeMBIによる強い誘導が認められるCYP1A1/2を選択した。ラットまたはヒトの肝細胞を4-MeMBI、5-MeMBIまたはMBIの存在下で48または96時間培養し、HPLC法を用いてtestosteroneの6β-ハイドロキシ化の測定によるCYP3A2/4活性、および7-ethoxyresorufinのO-脱エチル化の測定によるCYP1A1/2活性を調べた。
ラット肝細胞では、4-MeMBIおよび5-MeMBIはCYP3A2を阻害したが、4-MeMBIがCYP1A1/2を強く誘導した。MBIはCYP3A2を強く阻害し、CYP1A1/2を弱く阻害した。これらの結果には、報告されているin vivoの実験結果とは矛盾が認められず、4(5)-MeMBIの毒性に関する代謝的薬物間相互作用には、4-MeMBIおよび5-MeMBIを代謝するCYP1A1/2の4-MeMBIによる誘導が重要であると考えられた。
ヒト肝細胞では、ドナーに依存して、4-MeMBIおよび5-MeMBIによるCYP1AおよびCYP3A4の弱い誘導が認められた。これらの結果から、ヒトにおいては4(5)-MeMBIの毒性に関する代謝的薬物間相互作用が起きる可能性は低いことが示唆された。（Miyajima A *, Kuroda Y *, Sakemi-Hoshikawa K, Usami M, Mitsunaga K, Irie T, Ohno Y, Sunouchi M. Inhibitory and inductive effects of 4- or 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, on several forms of cytochrome P450 in primary cultured rat and human hepatocytes. Toxicology Reports. 2020. * These authors equally contributed to this work.）
5．ラット肝細胞における4-MeMBIおよび5-MeMBIのCYP mRNA誘導作用
mRNAレベルでCYP誘導効果を検討するために、4-MeMBIおよび5-MeMBIを8日間連続経口投与したラットにおけるCYP mRNAの変化率をCompetitive RT-PCR法を用いて調べた。その結果、4-MeMBIおよび5-MeMBIはCYPs 1A2、2B2、2C11、2E1および3A2のmRNA発現を誘導した。特にCYP1A2の誘導が大きく、4-MeMBIによる誘導量は5-MeMBIによる誘導量の８倍高かった。逆に、CYP2B2の誘導量は、5-MeMBIが4-MeMBIの２倍高かった。これらの結果から、in vivo実験およびin vitro実験における、CYPの活性増加は、mRNAのレベルでの発現誘導によると考えられた。（Yukie Kuroda*, Atsuko Miyajima*, Kazue Sakemi-Hoshikawa, Makoto Usami, Katsuyoshi Mitsunaga, Yasuo Ohno, and Momoko Sunouchi. Inducibility of cytochrome P450 (CYP) mRNAs by 4- or 5-methyl-2-mercaptobenzimidazole in the rat liver after repeated oral administration. ResearchGate. 2020. * These authors equally contributed to this work.）
6．総括
本研究では、in vitro実験系を用いて、4-MeMBIおよび5-MeMBIの細胞毒性、肝CYPによる代謝、そして肝CYPの誘導を調べた。その結果、4-MeMBI、5-MeMBIおよびMBIの細胞毒性の強さはMethyl-MBIs ＞ MBIだった。これはin vivoで報告されている毒性とは逆だった。4-MeMBIおよび5-MeMBIは、CYPs 1Aおよび2Bにより代謝されること、CYP1A活性を誘導することを示した。さらに、4-MeMBIおよび5-MeMBIによるCYPsの活性誘導はmRNAレベルでの発現誘導であることを示した。これらの結果から、4-MeMBIおよび5-MeMBIは、混合物4(5)-MeMBIとして用いられた場合に、肝CYPの発現誘導によりお互いの代謝を促進することにより甲状腺毒性および肝毒性を弱める、という代謝的薬物間相互作用を起こすと考えられた。これは、強い毒性を持つ化学物質を混合物として用いた場合には、代謝的薬物間相互作用により毒性を弱める、という有用な現象である。doctoral thesi
Postoperative improvement of megaesophagus and esophageal motility in a 19-day-old puppy with gastroesophageal intussusception
Get PDFCanine gastroesophageal intussusception (GEI) is a rare and life-threatening condition that requires prompt diagnosis and treatment. A 19-day-old Siberian Husky with a 4-day history of regurgitation was diagnosed with GEI based on the findings of computed tomography (CT) performed without anesthesia. Endoscopic reduction of intussusception was impossible; thus, surgical reduction by traction of the duodenum was performed. CT revealed improvement of megaesophagus 82 days postoperatively. Eleven months postoperatively, fluoroscopy showed recovery to nearly normal esophageal motility. Two years postoperatively, no clinical signs were reported. CT is useful to diagnose GEI in neonate puppies with poor abdominal fat and to assess the gastric edema and the anatomical association of stomach with other organs. Fluoroscopy is helpful for evaluating postoperative esophageal motility.journal articl
Promoter CpG methylation inhibits Krüppel-like factor 2 (KLF2)-Mediated repression of hTERT gene expression in human T-cells
Get PDFConstitutive expression of human telomerase reverse transcriptase (hTERT) with DNA methylation of its promoter is a common phenomenon in tumor cells. We recently found that the transcriptional factor Krüppel-like factor 2 (KLF2) binds to the CpG sequences in the hTERT promoter and inhibits hTERT gene expression in normal resting T-cells. The human T-cell line Kit 225 in the resting phase induced by the deprivation of interleukin (IL)-2 showed no decrease in the expression of hTERT, despite the high expression of KLF2. To elucidate the mechanisms of deregulation of hTERT expression in T-cells, we examined the relationship between DNA methylation and KLF2 binding to the hTERT promoter. The hTERT promoter was methylated in Kit 225 cells, resulting in the inhibition of the binding of KLF2 to the promoter. DNA demethylation by the reagent Zebularine recovered KLF2 binding to the hTERT promoter, followed by the downregulation of its gene expression. These findings indicate that the repressive effect of KLF2 on hTERT gene expression is abolished by DNA methylation in T-cell lines.journal articl
Investigation of the effects of excessive tibial plateau angle and changes in load on ligament tensile forces in the stifle joints of dogs
Get PDFObjective: To investigate the effect of an excessive tibial plateau angle (TPA) and change in compressive load on tensile forces experienced by the cranial cruciate, medial collateral, and lateral collateral ligaments (CCL, MCL, and LCL, respectively) of canine stifle joints.
Sample: 16 cadaveric stifle joints from 16 orthopedically normal Beagles.
Procedures: Stifle joints were categorized into unchanged (mean TPA, 30.4°) and excessive (mean TPA before and after modification, 31.2° and 41.1°, respectively) TPA groups. The excessive TPA group underwent a TPA-increasing procedure (curvilinear osteotomy of the proximal aspect of the tibia) to achieve the desired TPA. A robotic system was used to apply a 30- and 60-N compressive load to specimens. The craniomedial band of the CCL, caudolateral band of the CCL, MCL, and LCL were sequentially transected; load application was repeated after each transection. Orthogonal force components were measured in situ. Forces on ligaments were calculated after repeated output force measurements as the contribution of each component was eliminated.
Results: Increasing the compressive load increased tensile forces on the craniomedial and caudolateral bands of the CCL, but not on the MCL or LCL, in specimens of both groups. At the 60-N load, tensile force on the craniomedial band, but not other ligaments, was greater for the excessive TPA group than for the unchanged TPA group.
Conclusions and clinical relevance: Results indicated that stress on the CCL may increase when the compressive load increases. The TPA-increasing procedure resulted in increased tensile force on the CCL at a 60-N compressive load without affecting forces on the MCL or LCL.journal articl