{"created":"2023-06-19T07:19:18.742750+00:00","id":5343,"links":{},"metadata":{"_buckets":{"deposit":"a5b4d82e-5b47-4554-a6b1-b0a73016b47b"},"_deposit":{"created_by":4,"id":"5343","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"5343"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00005343","sets":["370:15:391"]},"author_link":["23015"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2019-03-15"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"32701","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"Chemical- or drug-related muscle toxicity, ranging from muscle pain to rhabdomyolysis, is induced by treatment with various compounds, including antilipidemic and hypocholesterolemic drugs, anesthetics, immunosuppressants, and proton pump inhibitors. Early detection of skeletal muscle toxicity is important because it has a critical influence on medical care and drug discovery, as skeletal muscle toxicity can develop into life-threatening rhabdomyolysis. Aspartate aminotransferase (AST) and creatine kinase (CK) are widely used as traditional biomarkers for skeletal muscle disorders. However, novel biomarkers that are superior to the conventional skeletal muscle biomarkers, AST and CK, or that can be combined with conventional biomarkers to compensate for the disadvantages of AST and CK are needed because AST and CK have low sensitivity and specificity. Several new biomarkers, including skeletal muscle troponin I (sTnI), myosin light chain 3 (Myl3), and fatty acid-binding protein 3 (FABP3), have been recommended for use with CK to monitor drug-induced skeletal muscle injury. However, several drawbacks are associated with the use of these biomarkers, including rapid clearance and alteration due to renal dysfunction. In this study, we performed an unbiased metabolomic analysis of skeletal muscle and plasma in a rat model of drug- and chemical-induced skeletal muscle injury to identify new candidate biomarkers with greater sensitivity than conventional biomarkers and examined the usefulness of the candidate biomarkers.\n\nChapter 1: Identification of New Biomarkers for Drug-Induced Skeletal Muscle Disorders Using Metabolomic Analysis\nTo identify new candidate biomarkers for skeletal muscle toxicity, an unbiased metabolomic analysis was performed in rats treated with two distinct myotoxicants, cerivastatin (CER) and tetramethyl-p-phenylenediamine (TMPD). Skeletal muscle toxicity was induced in male Fischer 344 rats through the administration of CER or TMPD and then monitored by using established endpoints, such as increased plasma creatine kinase (CK) activity and histopathology, and the metabolomic analysis of skeletal muscle and plasma samples. Plasma CK levels in CER-treated rats were markedly elevated on Day 11; however, those in TMPD-treated rats showed a statistically significant decrease at 24 hr after dosing. Light microscopy revealed the presence of vacuolated or necrotic fibers in all CER-treated rats on Day 11, with slightly vacuolated fibers observed in TMPD-treated rats at 6 and 24 hr after dosing. Metabolomic analysis of the rectus femoris indicated increases in 2-hydroxyglutarate (2HG) in CER-treated rats and hexanoylcarnitine in CER- and TMPD-treated rats. Increases in plasma 2HG were also observed in CER-treated rats on Days 8 and 11 and in TMPD-treated rats at 24 hr after dosing, and increases in plasma hexanoylcarnitine were observed in CER-treated rats on Day 11 and in TMPD-treated rats at 6 and 24 hr after dosing. These experiments demonstrated the potential of plasma 2HG and hexanoylcarnitine as specific and easily detectable biomarkers for skeletal muscle toxicity in rats and demonstrated the value of metabolomics for biomarker detection and identification in toxicological studies.\n\nChapter 2: A Study on the Usefulness of Plasma 2HG as a New Biomarker for Skeletal Muscle Disorder\nWe have demonstrated the potential of plasma 2HG as an easily detectable biomarker for skeletal muscle injury in rats. Therefore, we examined whether plasma 2HG was superior to conventional skeletal muscle damage biomarkers, including aspartate aminotransferase (AST), CK, and skeletal muscle-type CK isoenzyme (CK-MM) levels, in rats. Skeletal muscle injury was induced in 4- and 9-week-old male Fischer 344 rats by CER or TMPD administration. Plasma 2HG levels were measured on Days 4, 8, and 11 (CER group) and at 6 and 24 hr post administration (TMPD group). Plasma AST, CK, and CK-MM activities and histopathological changes in the rectus femoris muscle were evaluated as the study endpoints. In the CER group, AST, CK, and CK-MM increased in 4- and 9-week-old rats; in contrast, increases in CK (4- and 9-week-old rats) and CK-MM (4-week-old rats) were not obvious in the TMPD group. In both 4- and 9-week-old rats, plasma 2HG increased on Day 8 and at 24 hr post administration in the CER and TMPD groups, respectively. Histopathological analysis revealed myofiber vacuolation and necrosis in both groups. The histopathological damage to the rectus femoris muscle was more severe in the CER group than in the TMPD group. Increased plasma 2HG was associated with CER- and TMPD-induced skeletal muscle injuries in rats and was not affected by age differences or repeated blood collection. The results suggested that plasma 2HG was superior to CK and CK-MM as a biomarker for mild skeletal muscle injury.\n\nThe present study showed the elevation of 2HG and hexanoylcarnitine levels in muscle and plasma in a rat model of CER- and TMPD-induced skeletal muscle injury. Plasma 2HG increased prior to histopathological changes in skeletal muscle or the elevation of plasma CK in CER-treated rats. Further, the increases in plasma 2HG and hexanoylcarnitine were accompanied by histopathological changes, even when the plasma level of CK did not change. These results suggested that 2HG and hexanoylcarnitine may be novel plasma biomarkers for skeletal muscle damage. Subsequently, we measured the plasma concentrations of 2HG in rats with skeletal muscle injury and compared those with the levels of conventional biomarkers, such as AST, CK, and CK-MM, to evaluate the potential of plasma 2HG as a biomarker for skeletal muscle toxicity. Plasma 2HG was a more sensitive biomarker than CK and CK-MM in TMPD-induced mild skeletal myopathy. Furthermore, no differences were noted between younger and older rats or after repeated blood collection. These results suggested that plasma 2HG may serve as a promising novel biomarker for skeletal muscle toxicity.\nHowever, the organ or species specificity of plasma 2HG as a biomarker for muscle injury is not known. In addition, to establish 2HG as a biomarker for skeletal muscle disorders, it is necessary to perform a thorough comparison of plasma 2HG with other skeletal muscle injury biomarkers in terms of sensitivity, organ specificity, species difference and sex difference. The present study provided new and valuable evidence about 2HG as a biomarker for skeletal muscle disorders, and the results of this study will provide useful basic information for biomarker research for skeletal muscle disorder.\n","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"　薬剤の副作用の一つである骨格筋毒性は、軽度の筋肉痛から横紋筋融解症まで幅広くみられる。横紋筋融解症は生命に関わる可能性があることから、臨床あるいは医薬品開発の観点からも骨格筋毒性の早期検出が重要である。クレアチンキナーゼ（CK）とアスパラギン酸アミノトランスフェラーゼ（AST）は、骨格筋障害マーカーとして従来から広く用いられているが、CKおよびASTは溶血でも上昇する他、スタチンであるatorvastatinでは投与後に骨格筋機能に対する有害事象あるいは筋肉痛の発生頻度と関連しないCK上昇が発生する等、感度および特異性の低さが指摘されており、CKより感度および特異性の優れた新たな骨格筋障害マーカーが期待されている。Skeletal muscle troponin I（sTnI）、myosin light chain 3（Myl3）、およびfatty acid-binding protein 3（FABP3）は新規骨格筋障害マーカーとしてCKと併せて使用することが推奨されているが、血中からの消失の早さや測定値が腎機能の影響を受けるといった欠点が報告されている。本研究では、薬剤あるいは化学物質で骨格筋障害を誘発したラットの骨格筋および血漿のメタボロミクス解析を行い、従来の骨格筋障害バイオマーカーより高感度のマーカーを見出し、その有用性を評価することを目的とした。\n\n第1章　メタボロミクス解析を用いた薬物誘発骨格筋障害の新規バイオマーカーの探索\n　本章では、骨格筋障害の新たなバイオマーカー候補を見出すことを目的に、セリバスタチン（CER）あるいはtetramethyl-p-phenylenediamine（TMPD）を投与して骨格筋障害を誘発したラットを用いてメタボロミクス解析を行った。CER（0あるいは40 ppm、混餌投与）あるいはTMPD（0あるいは9 mg/kg、単回経口投与）を9週齢の雄性Fischer 344（F344）ラットに投与して骨格筋障害を誘発し、血漿CK測定および骨格筋の病理組織学的解析により骨格筋毒性を評価するとともに、骨格筋（大腿直筋）および血漿について、内因性代謝物を網羅的に測定するメタボロミクス解析を行い、CER群およびTMPD群の骨格筋および血漿中で共通して増加した代謝物を調べた。CER群の血漿CKはDay 11に顕著に上昇したが、TMPD群の血漿CKは投与24時間後に有意に低下した。病理組織学的検査では、CER群のDay 11に骨格筋細胞の空胞化および壊死を認め、TMPD群の投与6および24時間後に骨格筋細胞の軽度の空胞化を認めた。メタボロミクス解析では、大腿直筋で2-hydroxyglutarate（2HG）がCER群で、hexanoylcarnitine がCER群およびTMPD群で増加した。血漿では、2HGがCER群のDay 8および11、TMPD群の投与24時間後に増加し、hexanoylcarnitineがCER群のDay 11、TMPD群の投与6および24時間後に増加した。以上の結果から、血漿中2HGおよびhexanoylcarnitineは、ラットの骨格筋障害の初期段階における新たな骨格筋障害マーカーとなる可能性が示唆された。\n第2章　新規骨格筋障害バイオマーカーとしての血漿中2HG濃度の有用性検討\n　本章では、バイオマーカーとしての血漿中2HG濃度の有用性を調べることを目的に、血漿2HGの骨格筋障害に対する感度を、従来からの骨格筋障害バイオマーカーであるAST、CK、および骨格筋型CKアイソザイム（CK-MM）と比較し、週齢差、および反復採血の影響について検討した。4あるいは9週齢の雄性Fischer 344ラットにCERを0あるいは20 ppm（4週齢）、40 ppm（9週齢）を10日間混餌投与した。また、TMPDを0あるいは9 mg/kg（4および9週齢）を単回経口投与することにより骨格筋障害を誘発した。経時的（CER群はDay 4、8、11、TMPD群は投与6、24時間後）に採血し、血漿中2HG濃度をLC-MS/MSで測定した。頚静脈からの最終採血後に剖検し、血漿中AST、CK、CK-MM活性の測定、ならびに骨格筋（大腿直筋）の病理組織学的解析を行った。CER群ではAST、CK、CK-MMは両週齢で増加したが、TMPD群ではCK（4および9週齢）、CK-MM（4週齢）の増加は認められなかった。2HGはCER群ではDay 8（4および9週齢）から、TMPD群では投与6時間後以降（4週齢）あるいは24時間後（9週齢）に増加した。病理組織学的検査では、CER群およびTMPD群の両週齢で骨格筋細胞の空胞化または壊死がみられ、病理組織学的変化の程度はTMPD群ではCER群と比較して軽度であった。CERあるいはTMPDで誘発した骨格筋障害に伴う血漿中2HG濃度の増加について、週齢差および反復採血の影響は認められなかった。以上の結果から、血漿中2HGは骨格筋障害の初期段階においてCKおよびCK-MMより高感度であること、さらに週齢差および反復採血の影響が認められないことが示され、骨格筋毒性評価に有用である可能性が示唆された。\n\n　本研究では、CERあるいはTMPDで骨格筋障害を誘発したラットの骨格筋および血漿中で2HGおよびhexanoylcarnitineが増加することが明らかとなった。血漿2HGはCER群の骨格筋の病理組織学的変化または血漿CKの増加に先んじて増加した。さらに、血漿2HGおよびhexanoylcarnitineはTMPD群の病理組織学的変化に伴って増加したが、血漿CKは増加しなかった。これらの結果から、2HGおよびhexanoylcarnitineが骨格筋障害の新たなバイオマーカー候補である可能性が示唆された。血漿中2HGのバイオマーカーとしての有用性を検討するために、血漿2HGと従来の骨格筋障害マーカーであるAST、CK、CK-MMを比較した結果、TMPDによる軽度の骨格筋障害において、血漿2HGはCKおよびCK-MMよりも高感度であった。また、週齢差および反復採血の影響がみられなかった。これらの結果から、血漿2HGが骨格筋毒性の有望な新規バイオマーカーとして有用であることが示唆されたが、血漿2HGの骨格筋障害マーカーとしての臓器および種特異性は不明である。また、2HGを骨格筋障害のバイオマーカーとして確立するには、他の骨格筋障害マーカーとの比較と背景データの取得が必要であり、今後の研究が望まれる。しかしながら、本研究はバイオマーカー候補としての2HGの新たな知見を示すとともに、骨格筋障害を早期に検出するバイオマーカー研究のための有用な基礎情報になると考える。","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第153号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"大林, 久佐邦"}],"nameIdentifiers":[{"nameIdentifier":"23015","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-03-22"}],"displaytype":"detail","filename":"diss_dv_kou0153.pdf","filesize":[{"value":"5.4 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_kou0153","url":"https://az.repo.nii.ac.jp/record/5343/files/diss_dv_kou0153.pdf"},"version_id":"fa43ce99-e97f-4629-9b7f-7f0a243a93b3"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-03-22"}],"displaytype":"detail","filename":"diss_dv_kou0153_jab&rev.pdf","filesize":[{"value":"195.5 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_kou0153_jab&rev.pdf","url":"https://az.repo.nii.ac.jp/record/5343/files/diss_dv_kou0153_jab&rev.pdf"},"version_id":"b2db4c19-d659-4118-95da-06ec98d4f9d8"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis","resourceuri":"http://purl.org/coar/resource_type/c_db06"}]},"item_title":"薬物誘発性骨格筋障害の新規バイオマーカー探索に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"薬物誘発性骨格筋障害の新規バイオマーカー探索に関する研究"},{"subitem_title":"Biomarker exploration for drug-induced skeletal muscle disorders","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["391"],"pubdate":{"attribute_name":"公開日","attribute_value":"2019-04-23"},"publish_date":"2019-04-23","publish_status":"0","recid":"5343","relation_version_is_last":true,"title":["薬物誘発性骨格筋障害の新規バイオマーカー探索に関する研究"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T07:41:16.831696+00:00"}