Toxicological studies of citreoviridin that is a mycotoxin contaminating rice and grains

シトレオビリジン（CTVD）は、Penicillium citreonigrum、Aspergillus terreus、Eupenicillium ochrosalmoneumなどが二次代謝産物として産生するカビ毒である。CTVDを産生するP. citreonigrumなどは穀類のうち主にコメを汚染するため、コメを主食とするアジアや南米などの国々でCTVDの汚染は問題となる。CTVDに汚染されたコメが黄色くなることから、CTVDは黄変米毒の一つとしても知られており、日本国内で過去に輸入米での汚染事例が報告されている。近年においても、タイやブラジルなど熱帯性気候の地域でCTVD産生能を有するP. citreonigrumの存在が報告されている。　CTVDの毒性については、1940年にP. citreonigrumに汚染されたコメの粗抽出物を腹腔内投与（IP）、皮下投与（SC）又は経口投与（PO）により哺乳類や脊椎動物に投与した場合に、四肢の進行性麻痺、嘔吐、痙攣、漸次的呼吸障害などを引き起こすことがいくつかの毒性学的研究から明らかとなった。その原因カビ毒としてCTVDが同定され、精製品を用いた動物実験において、神経症状の進行時間は毒素抽出物の用量が多くなるほど短くなることが報告されている。マウスに対するCTVDのLD50は3.6-11.8 mg/kg（皮下投与）、7.5 mg/kg（腹腔内投与）とされている。ヒトで起こる健康被害としては、衝心脚気やKeshan病との関連も指摘されており、2006年にブラジルで発生した衝心脚気のアウトブレイクでは、当該地域のコメからCTVDを産生するP. citreonigrumが検出されており、同毒素が原因物質として疑われている。　毒性学研究においてはトキシコキネティクスや体内動態の情報が欠かせないが 、CTVDの毒性実験が盛んに行われていた1940年代から1980年代には微量分析が可能な分析機器等がなく、感度の低い薄層クロマトグラフィーによる蛍光分析での結果に留まっていた。そこで、本研究ではCTVDのトキシコキネティクスを明らかにする目的で、解剖学的、生理学的にヒトと類似しているブタを用いたCTVDの投与実験により検討した。また、ヒトとの比較を行うため、ヒト腸管細胞モデルであるCaco-2細胞を用いた透過実験及び代謝酵素を含む肝臓S9画分を用いた代謝実験をin vitroで行い、ヒトにおけるCTVDのバイオアベイラビリティを推測した。本研究の概要は次のとおりである。Ⅰ ブタにおけるCTVDのトキシコキネティクス　約10 kgのブタにCTVD 0.1 mg/kg bwを静脈投与及び経口投与した。継時的に血液を採取し、分離した血漿中のCTVD濃度をLC-MS/MSで分析した。分析結果から解析ソフト(WinNonlin)を用いてトキシコキネティクスパラメータ(TKパラメータ)を算出し、次の結果を得た。（１）静脈投与後のCTVDの血漿中濃度は、投与後急激に減少し、最終採血時点である48時間後まで緩やかに減少したが48時間後でも血漿中からCTVDが検出された。（２）静脈投与時のTKパラメータは、小さな速度定数（Kel）（0.5 ± 0.1 ×10-1 h-1）を示し、半減期（T1/2）及び分布容積（Vd）はそれぞれ16.2 ± 4.3 h、1.5 ± 0.2 Lであった。（３）経口投与後のCTVDの血漿中濃度は、投与後緩やかに上昇し、15.0 ± 6.0 h（Tmax）でピーク（Cmax：38.2 ±6.7 ng/mL）を迎えたのち、48時間後まで緩やかに減少した。（４）経口投与時のTKパラメータは、静脈投与と同様にKelが小さく（0.4±0.2 ×10-1 h-1）、比較的長いT1/2（21.4 ± 12.7 h）と大きなVd（1.7 ± 0.3 L）を示した。（５）CTVDのブタにおけるバイオアベイラビリティは、投与後48時間までで79.3 %と比較的高く、無限時間まで外挿した場合には116.4%を示した。これらの結果から、CTVDはブタにおいて高いバイオアベイラビリティを持つことが示唆された。また、CTVDは体内からの消失が遅く、組織移行性も高いと考えられ、比較的長く体内に残留することが示唆された。Ⅱ ヒト腸管培養細胞Caco-2細胞を用いたCTVDの透過係数　ヒトにおけるCTVDのバイオアベイラビリティを推定するため、ヒト腸管細胞モデルであるCaco-2細胞を用いた透過実験を実施した。CorningTM BioCoatTM Intestinal Epithelium Differentiation Environment Kitを用い、単層に培養したCaco-2細胞の粘膜面側にCTVD（3 µM及び10 µM）を暴露し、37℃でインキュベートした。2時間後の粘膜面側及び基底膜面側の培地を採取し、培地中のCTVD濃度をLC-MS/MSを用いて測定して、経上皮電気抵抗値（TEER）及び透過係数（Papp）を求めた。その結果、（１）いずれのCTVD濃度においてもTEERに変化は認められなかった。（２）各CTVD濃度におけるPappは、それぞれ52.2 ± 28.3、42.6 ± 17.7 （×10-6 cm/s）であり、比較的高い透過係数を示した。これらから、CTVDはブタと同様にヒトにおいても体内に吸収されやすいことが示唆された。高い透過係数は、CTVDの脂溶性の高さを反映する結果と考えられる。Ⅲ ブタ及びヒト肝臓S9画分を用いたCTVDの代謝とその代謝物　ブタを用いたCTVDのin vivo実験において、血漿中CTVD濃度が比較的緩やかに減少し、48時間後においても血漿中に残留していたことから、ブタにおいては代謝が比較的遅い可能性が示唆された。そのため代謝酵素を含むブタ及びヒトの肝臓S9画分を用いた代謝実験によりCTVDへの代謝能を比較した。始めにグルコース−６−リン酸（G-6-P）及びNADPを含む溶液中に肝臓S9画分（0.5 mg/mL）及びCTVD（1.5 µg/mL）を加え、37℃でインキュベートした。インキュベート後30分、1時間及び4時間後の溶液を採取し、Q-TOFで分析した。その結果、（１）CTVDの代謝物は主に水酸化-メチル化体、不飽和化体及びジヒドロキシ化体であることが推測された。（２）いずれの代謝物もヒト肝臓S9画分を用いたほうがブタ肝臓S9画分を用いるよりも有意に多く産生された。（３）CTVD濃度がブタ肝臓S9画分を用いるよりもヒト肝臓S9画分を用いたほうが有意に低下した。これらから、CTVDは、ブタにおいてS9による代謝が遅いことが示唆され、ヒトにおいてはブタよりもCTVDを代謝しやすいことが考えられた。　S9画分を用いた代謝実験において、グルクロン酸抱合体が産生されなかったことから、ウリジン二リン酸グルクロン酸及びアラメチシンの存在下でCTVDとS9画分をインキュベートして、CTVDのグルクロン酸抱合体の産生を調べた。その結果、（１）いずれのS9画分を用いた場合でも、インキュベート後30分までにCTVDのグルクロン酸抱合体は産生されなかった。（２）インキュベート後4時間でブタ肝臓S9画分を用いたほうがヒト肝臓S9を用いるよりも有意にグルクロン酸抱合体が産生された。これらから、CTVDのグルクロン酸抱合体についてはブタのほうがヒトよりも産生されやすいことが示唆された。　本研究では、in vivoにおけるブタでのCTVDのトキシコキネティクスの結果より、CTVDが体内に吸収されやすく、長い半減期と大きい分布容積を持つことが示された。これらのことから比較的長く体内に残留することを明らかにした。また、CTVDのバイオアベイラビリティはブタにおいて高いだけでなく、ヒトにおいても高い可能性をin vitroで明らかにした。一方で、CTVDの代謝については、ヒトではブタよりも早いが、グルクロン酸抱合体形成能はブタのほうが優れていたことをin vitroの観点から明らかにした。Citreoviridin (CTVD) is a mycotoxin that is a secondary metabolite produced by Penicillium citreonigrum, Aspergillus terreus and Eupenicillium ochrosalmoneum. Because P. citreonigrum capable of producing CTVD contaminates mainly rice, contamination with CTVD can be problem in countries of Asia and South America, where rice is a staple food. CTVD is a yellow rice toxin, and a case of its contamination in an imported rice sample was previously reported in Japan. Recently, the occurrence of P. citreonigrum capable of producing CTVD has been reported in areas with a tropical climate, such as Thailand and Brazil.Regarding toxicity of CTVD, in the 1940s, several toxicological studies revealed that symptoms such as progressive paralysis, vomiting, convulsions and temporary respiratory disturbance developed when a crude extract from contaminated rice was administered to mammals and other vertebrates via intraperitoneal (IP), subcutaneous (SC) and oral (PO) routes. Furthermore, it was found that isolated CTVD causes the same symptoms as experiments using moldy rice. Neurological symptoms progressed faster at higher doses of extracted toxin, and the median lethal dose (LD50) of CTVD for mice has been reported be 3.6 to 11.8 mg/kg (SC) and 7.5 mg/kg (IP). As a human adverse effects, CTVD has been considered to be related to Shoshin-kakke (acute cardiac beriberi) and Keshan disease which adversely affects the heart. In 2006, an outbreak of Shoshin-kakke occurred in Brazil, and the presence of rice contaminated with P. citreonigrum capable of producing CTVD was reported in the outbreak area.An understanding of toxicokinetics is essential when performing a toxicological study. However, from the 1940s to the 1980s, when toxicological studies of CTVD were performed actively, no high-resolution analytical methods had yet been developed, and the toxicokinetics of CTVD were determined based solely on the results of a fluorescence analysis using thin layer chromatography.The present study was conducted with the following three experiments: (1) the toxicokinetics of CTVD were investigated by administering CTVD to swine, which are similar to humans both anatomically and physiologically; (2) the permeability of CTVD was investigated using Caco-2 cells, which are a model of human intestinal cells; and (3) a CTVD metabolic study was conducted using the swine or human hepatic S9 fraction containing metabolic enzymes. The bioavailability of CTVD to humans was estimated, while in addition the toxicokinetics of CTVD in vitro to humans was compared with that to swine on studies (2) and (3). I. Toxicokinetics of CTVD in swineCTVD (0.1 mg/kg body weight) was administered to swine (weighing about 10 kg) intravenously (IV) and PO. The CTVD concentration of plasma samples was determined via liquid chromatography with tandem mass spectrometry (LC-MS/MS). The toxicokinetics parameters (TK) were analyzed, and the following results were obtained: (1) the CTVD concentration in plasma decreased quickly shortly after IV administration and then continued to decrease moderately until 48 h (final sampling point); however, some amount of CTVD remained in plasma even at 48 h after administration. (2) The rate constant (Kel) after IV administration was 0.5 ± 0.1 × 10 -1 h-1. The half-life (T1/2) and volume of distribution (Vd) were 16.2 ± 4.3 h and 1.5 ± 0.2 L, respectively. (3) The CTVD concentration in plasma in PO gently increased after administration and reached the maximum concentration (Cmax: 38.2 ± 6.7 ug/mL) at 15.0 ± 6.0 h (Tmax), after which it moderately decreased until 48 h. (4) The Kel after PO administration was small (0.4 ± 0.2 × 10-1 h-1, similar to that in IV). The T1/2 and Vd were 21.4 ± 12.7 h and 1.7 ± 0.3 L, respectively. (5) The bioavailability of CTVD was 79.3% from 0 to 48 h and 116.4% on extrapolating from 48 h to infinity. These results suggested that CTVD had a high bioavailability in swine. In addition, CTVD was eliminated slowly from the body and was easily distributed to tissues. These findings suggested that CTVD remained in the body for a relatively long time after administration.II. Permeability of CTVD for Caco-2 cells, a human intestinal cell modelBecause CTVD had a relatively high bioavailability in swine, a permeability study was performed using Caco-2 cells, which are a human intestinal cell model. CTVD (3 μM and 10 μM) was treated to the apical side (AP) of Caco-2 cells cultured in a monolayer using a CorningTM BioCoatTM Intestinal Epithelium Differentiation Environment Kit (Corning, NY, USA) and then incubated at 37 °C. After 2 h, the culture medium was sampled from the AP and basolateral side (BL). The CTVD concentration was determined using LC-MS/MS, and then the transepithelial electrical resistance (TEER) and permeability coefficient (PaPP) were estimated. The results of this study were as follows: (1) The TEER was unchanged at each concentration of CTVD. (2) The PaPP values at each CTVD concentration of 3 μM and 10 μM were 52.2 ±28.3 and 42.6 ± 17.7 (×10-6 cm/s), respectively, indicating a relatively high permeability. These findings suggested that CTVD could be easily absorbed by the human intestine, similar to swine, which reflected a high lipid solubility of CTVD.III. CTVD metabolism and its metabolites as assessed using swine and human hepatic S9 fractionsOur in vivo study showed that the plasma CTVD concentration decreased gently, and CTVD was still detected even at 48 h after administration. Given these results, the CTVD metabolism was next investigated using swine and human hepatic S9 fractions containing metabolic enzymes. CTVD was incubated at 37 °C with hepatic S9 fraction in solution containing glucose-6-phosphate (G6P) and NADP. The solution was sampled at 30 min, 1 and 4 h after incubation, and then the concentrations of CTVD and its metabolites were determined using a quadrupole time-of-flight mass spectrometer (Q-TOF). The findings were as follows: (1) The CTVD metabolites were estimated to be hydroxylation-methylation, desaturation and dihydoroxylation derivatives. (2) Each metabolite was produced in greater quantities using the human hepatic S9 fraction than the swine hepatic S9 fraction. (3) The CTVD concentration was significantly lower with the human hepatic S9 fraction than with the swine hepatic S9 fraction. These results suggested that CTVD was slowly metabolized in swine, which may have been the cause of the slow elimination rate noted in the swine in the in vivo study. In addition, humans seemed to more readily metabolize CTVD than swine.Because CTVD glucuronide was unable to be detected in the metabolic study using S9 fractions in the presence of G6P and NADP, CTVD was incubated with an S9 fraction in the presence of UDP-glucuronic acid and alamethicin to examine CTVD glucuronidation ability. The following results were obtained: (1) CTVD glucuronide had not been produced by 30 min after incubation, regardless of the fraction used. (2) The levels of CTVD glucuronide at 4 h after incubation were significantly greater with the swine hepatic S9 fraction than with the human hepatic S9 fraction. These findings suggested that CTVD glucuronide was more readily produced in swine than in humans.The present studies showed that CTVD is easily absorbed by the body and has a long T1/2 and large Vd, indicating that CTVD persists for a relatively long time in the body. In addition, the bioavailability of CTVD appeared to be high in both humans and swine. Regarding CTVD metabolism, humans were able to metabolize CTVD earlier than swine according to in vitro findings, while the glucuronidation ability for CTVD was better in swine than in human from an in vitro perspective.博士(学術)麻布大

Instability of Population III Black Hole Accretion Disks

We investigate the stability of black hole accretion disks in a primordial environment (POP III disks for short), by solving the vertical structure of optically thick disks, including convective energy transport, and by employing a one-zone model for optically thin isothermal disks. Because of the absence of metals in POP III disks, we find significant differences in stability associated with ionization between POP III disks and the disks of solar metallicity. An unstable branch in S-shaped equilibrium curves on the Mdot-Sigma (mass accretion rate - surface density) plane extends to a larger surface density compared with the case of disks of solar metallicity. The resulting equilibrium loci indicate that quasi-periodic oscillations in luminosity can also be driven in POP III disks, and their maximal luminosity is typically by an order of magnitude larger than that of the disks of solar metallicity. Such a strong outburst of POP III disks can be observed by future huge telescopes, in case that the mass is supplied onto the disks at the Bondi accretion rates in typical virialized small dark halos.Comment: 7 pages, 4 figures, accepted for publication in PAS

Association Between Eosinophilia and Late-onset Circulatory Collapse in Preterm Infants: A case-Control Study

Late-onset circulatory collapse (LCC) in preterm infants is presumably caused by relative adrenal insufficiency. Because eosinophilia is known to be associated with adrenal insufficiency, we attempted to clarify the relation-ship between eosinophilia and LCC in preterm infants. We divided the cases of the infants (born at < 28 weeks’ gestation) admitted to our neonatal intensive care unit in 2008-2010 into 2 groups: those diagnosed with LCC that received glucocorticoids (LCC group), and those who did not receive glucocorticoids (control group). We compared eosinophil counts between the 2 groups and between before and after glucocorticoid treatment in the LCC group. A total of 28 infants were examined: LCC group (n = 12); control group (n = 16). The peak eosin-ophil counts of the LCC group were significantly higher than those of the control group (median: 1.392 × 109/L vs. 1.033 × 109/L, respectively; p = 0.02). Additionally, in the LCC group, the eosinophil counts declined significantly after glucocorticoid treatment (0.877 × 109/L vs. 0.271 × 109/L, p = 0.003). Eosinophil counts in the LCC group were significantly higher than in the control group and decreased rapidly after gluco-corticoid treatment. These results indicate that eosinophilia may be a factor associated with LCC caused by adrenal insufficiency

Gig workers, social protection and labour market inequality: lessons from Malaysia

The rise of the Gig Economy has created new concerns about inequality in rapidly digitising Asian economies. Whilst the emergence of the “on-demand economy” creates new opportunities for the youths and low-income groups, this has also created new policy challenges. As many use unofficial digital platforms and/or operate as unregistered self-run businesses, they remain outside the social protection system, leaving them especially vulnerable during times of crisis. This study therefore aims to provide new insights into this issue - social protection for gig workers – drawing upon the Malaysian experience. Using primary data collected through semi-structured interviews, we examine the working conditions of e-hailing drivers and online food delivery riders, two of the largest mainstream on-demand appbased gig workers’ groups in the country. The collected data were examined based on thematic analysis employing 15 subtopics and 3 main themes: (i) performance-based short-term decent work; (ii) platform and customer-centricity operations, and (iii) ecosystem for gig worker sustainability. From the perspective of gig workers, the study narrates their actual working conditions and perceived inequalities. Based on the findings, we conclude by critically reflecting on the potential for developing a worker-centred policy for social protection in the Gig Economy in Malaysia and elsewhere in Asia

Is the number of covered intercostal arteries a predictor of postoperative spinal cord ischemia after thoracic endovascular aortic repair?

Objective: The purpose of this study was to investigate the impact of the number of covered intercostal arteries (ICAs) on postoperative spinal cord ischemia (SCI) after Thoracic endovascular aortic repair (TEVAR).Methods: A retrospective review of a collected database was performed for all patients who underwent TEVAR at the Sapporo Medical University between January 2006 and February 2016. The pre- and post-operative thin slice contrast-enhanced computed tomography was performed, and ICAs were evaluated. Preoperative demographics, procedure-related variables, and clinical details related to SCI were examined. Logistic regression analysis was performed to identify risk factors for the development of SCI.Results: Of the 263 patients who underwent TEVAR during the study period, 11 patients (4.1%) developed SCI. The number of patent preoperative ICAs was 10.1 ± 4.4. There was no significant difference in the number of patent ICAs between the SCI and No SCI groups. On the other hand, the number of postoperative covered ICAs was 4.8 ± 3.3. The number of covered ICAs was higher in the SCI than No SCI group (8.3 ± 2.9 vs 4.7 ± 3.2, p = 0.001). The cut-off value was set at 6 ICAs by ROC curve analysis. Multivariate analysis demonstrated that in TEVAR, the covering of 6 or more ICAs by stent grafts became a significant risk factor for SCI (odds ratio, 10.9; p = 0.029).Conclusions: The number of covered ICAs becomes a predictor of postoperative SCI after TEVAR. The patient with 6 or more ICAs covered by stent grafts is deemed to require a more careful perioperative management

Labour Union’s challenges for improving for gig work conditions on food delivery in Japan: a lesson for Malaysia

The development of technology and the flexibility to work in nontraditional ways has led to the emergence of task-based gig workers and this trend has been spreading across Japan. However, the ambiguity of the legal position on app-based on-demand gig workers has led to inequalities in labour relations. This study examines the labour union actions against the employers and related bodies of the online food delivery platform Uber Eats Union to determine the current status and potential for bargaining. The union has requested collective bargaining on the Uber side, but all requests have been rejected. The admissibility of the union’s actions to improve industrial relations depended on two points: the application of the worker character in the law to the delivery workers and the demand for support from other institutions. In terms of the law, the recognition of the worker status of delivery workers is a significant step towards collective bargaining with firms. The active work of the Uber Eats Union could lead to the formation of new labour unions on other platforms and the empowerment of platform workers. Based on Japan’s experience, this paper also draws some lessons for Malaysia’s gig economy