Akiko Inomata

The multiwalled carbon nanotube, MWCNT-7, is carcinogenic to rat lungs when inhaled over the course of 2 years, but does not induce pleural mesothelioma. In contrast, intratracheal instillation of MWCNT-7 causes both lung tumors and pleural mesotheliomas. To explore the possibility of the use of the intratracheal instillation technique in risk characterization, we administered this widely used reference material using instillation to male F344 rats at doses of 0.0175, 0.07, 0.28, or 0.42 mg/kg once per week for 13 weeks, followed by an observation period of 91 weeks and compared the dose–response toxicological data with the 2-year inhalation study. Lung tumors and pleural mesotheliomas were induced in a dose-dependent manner (the 0.42 mg/kg group was excluded from analysis of lung tumor formation). The lung burden of MWCNT-7 at week 13 was dose-dependent: 0.0302, 0.191, 0.98, and 1.369 mg/lung in the 0.0175, 0.07, 0.28, and 0.42 mg/kg groups, respectively. The number of fibers in the pleural lavage fluid was also dose-dependent. The no-observed-adverse-effect-level (NOAEL) of MWCNT-7’s carcinogenicity was 0.0302 mg/lung based on no significant lung tumor induction and no mesothelioma induction in the 0.0175 mg/kg group. When compared based on lung burden, the dose–response relationship and NOAEL of lung tumor induction resembled those in the 2-year inhalation study, offering the possibility of the application of this protocol for risk characterization.

Despite intensive toxicological studies of carbon nanotubes (CNTs) over the last two decades, only a few studies have demonstrated their pulmonary carcinogenicities in chronic animal experiments, and the underlying molecular mechanisms are still unclear. To obtain molecular insights into CNT-induced lung carcinogenicity, we performed a transcriptomic analysis using a set of lung tissues collected from rats in a 2-year study, in which lung tumors were induced by repeated intratracheal instillations of a multiwalled carbon nanotube, MWNT-7. The RNA-seq-based transcriptome identified a large number of significantly differentially expressed genes at Year 0.5, Year 1, and Year 2. Ingenuity Pathway Analysis revealed that macrophage-elicited signaling pathways such as phagocytosis, acute phase response, and Toll-like receptor signaling were activated throughout the experimental period. At Year 2, cancer-related pathways including ERBB signaling and some axonal guidance signaling pathways such as EphB4 signaling were perturbed. qRT-PCR and immunohistochemistry indicated that several key molecules such as Osteopontin/Spp1, Hmox1, Mmp12, and ERBB2 were markedly altered and/or localized in the preneoplastic lesions, suggesting their participation in the induction of lung cancer. Our findings support a scenario of inflammation-induced carcinogenesis and contribute to a better understanding of the molecular mechanism of MWCNT carcinogenicity.

本研究では、リアルタイムRT-PCR法を用いた河川水中のクリプトスポリジウムオーシストの高感度定量法を開発した。はじめに、これまでに報告された複数のリアルタイムPCR系に着目し、感度や特異性等の比較を行った。Millerらによって報告されたPCR系は非特異産物を生成せず、感度も良好であったため、最も実用的であると考えられた。次に、Millerらによって報告されたリアルタイムPCR系に逆転写反応を組み合わせたリアルタイムRT-PCR法を構築し、定量感度の向上を試みた。逆転写反応によって、1オーシストあたり約27,000コピーのcDNAが生成されることがわかり、またリアルタイムRT-PCR法の定量下限は7.5×10－4 oocysts/tubeと判断され、極めて低濃度のクリプトスポリジウムオーシスト由来の核酸を定量可能であることがわかった。最後に、構築したリアルタイムRT-PCR法を水環境サンプル中のクリプトスポリジウム濃度の測定に適用し、顕微鏡観察を基本とする従来法と比較した。その結果、新しく構築したリアルタイムRT-PCR法は、特異性の問題を生じることなく、実河川試料からクリプトスポリジウムオーシストを定量することが可能であり、顕微鏡観察による推定オーシスト数と同様の結果が得られた。

We describe a novel assay for simple, rapid and high-sensitive detection of Cryptosporidium oocysts in water samples using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The detection limit of the developed RT-LAMP assay was as low as 6 x 10(-3) oocysts/test tube, which theoretically enables us to detect a Cryptosporidium oocyst and perform duplicated tests even if water samples contain only one oocyst. The developed RT-LAMP assay could more sensitively detect Cryptosporidium oocysts in real water samples than the conventional assay based on microscopic observation.

Heterotrophic plate count (HPC) test has been employed to indicate the effectiveness of water treatment processes and the microbiological condition of the distribution system. In Japan, because the majority of HPC bacteria are supposed to be harmless and all tap water should maintain residual chlorine, there are few studies of the virulence of HPC bacteria. In this study, we examined HPC bacteria isolated from finished and tap water for hemolytic activity to determine their virulence potential. 34 of 39 colonies expressing hemolytic activity were identified by partial 16S rDNA sequencing, but some of their percent identity were relatively low. This may have been due to the mismatching of the primer pair with some strains, or these strains may be unidentified new species. A total of 30 of 34 isolates identified have been reported to be opportunistic pathogens or food poisoning bacteria. To control the growth of these opportunistic pathogens among HPC bacteria, appropriate water quality control must always be done and residual chlorine must be maintained in every tap for a safe water supply.