Publications by authors named "Yasumasa Okazaki"

54 Publications

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide.

Arch Biochem Biophys 2021 Jul 6;705:108901. Epub 2021 May 6.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (OH), hydrogen peroxide (HO), singlet oxygen (O), superoxide (O), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); O using a fluorescent probe; and O and HO using luminescent probes in the presence of thiols or tempol. NTP-induced OH was significantly scavenged by dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) in 2 or 5 mM DMPO. NTP-induced O was significantly scavenged by 10 μM DTT and GSH, while O was not efficiently scavenged by these compounds. GSSG degraded HO more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with HO. In the presence of 1-50 mM DMPO, NTP-induced HO quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on HO production was observed in 1 and 10 mM DMPO, whereas it became ineffective in 50 mM DMPO. Furthermore, DMPO-OH did not interact with tempol. These results suggest that DMPO and tempol react competitively with O. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.
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http://dx.doi.org/10.1016/j.abb.2021.108901DOI Listing
July 2021

The therapeutic potential of curcumin in alleviating N-diethylnitrosamine and iron nitrilotriacetate induced renal cell tumours in mice via inhibition of oxidative stress: Implications for cancer chemoprevention.

Biomed Pharmacother 2021 Jul 3;139:111636. Epub 2021 May 3.

Department of Pathological Research, Faculty of Medicine, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-Cho, Okayama 700-8558, Japan.

This study was designed to reveal the protective effects of dietary supplementation of curcumin against renal cell tumours and oxidative stress induced by renal carcinogen iron nitrilotriacetate (Fe-NTA) in ddY male mice. The results showed that mice treated with a renal carcinogen, Fe-NTA, a 35% renal cell tumour incidence was noticed, whereas renal cell tumour occurrence was elevated to 80% in Fe-NTA promoted and N-diethylnitrosamine (DEN)-initiated mice as compared with saline- treated mice. No incidence of tumours has been observed in DEN-initiated non-promoted mice. Diet complemented with 0.5% and 1.0% curcumin fed prior to, during and after treatment with Fe-NTA in DEN-initiated animals, tumour incidence was reduced dose-dependently to about 45% and 30% respectively. Immunohistochemical studies also revealed the increased formation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in kidney tissue of mice treated with an intraperitoneal injection of Fe-NTA (6.0 mg Fe/kg body weight.). Furthermore, Fe-NTA treatment of mice also resulted in significant elevation of malondialdehyde (MDA), serum urea, and creatinine and decreases renal glutathione. However, the changes in most of these parameters were attenuated dose-dependently by prophylactic treatment of animals with 0.5% and 1% curcumin diet, this may be due to its antioxidative impact of curcumin. These results suggest that intake of curcumin is beneficial for the prevention of renal cell tumours and oxidative stress damage mediated by renal carcinogen, Fe-NTA.
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http://dx.doi.org/10.1016/j.biopha.2021.111636DOI Listing
July 2021

L-Dehydroascorbate efficiently degrades non-thermal plasma-induced hydrogen peroxide.

Arch Biochem Biophys 2021 03 20;700:108762. Epub 2021 Jan 20.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

Non-thermal plasma (NTP) devices generate reactive oxygen species (ROS) and reactive nitrogen species, such as singlet oxygen (O), superoxide (O), hydroxyl radical (OH), hydrogen peroxide (HO), ozone, and nitric oxide at near-physiological temperature. In preclinical studies, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of ROS in the liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here, we utilized electron paramagnetic resonance spectroscopy to quantitate OH, using a spin-trapping probe 5,5-dimethyl-1-pyrroline-N-oxide; O, using a fluorescent probe; and O and HO, using luminescent probes, after NTP exposure in the presence of antioxidants. l-ascorbate (Asc) at 50 μM concentration (physiological concentration in serum) significantly scavenged OH, whereas (-)-epigallocatechin gallate (EGCG) and α-tocopherol were also effective at performing scavenging activities at 250 μM concentrations. Asc significantly scavenged O and HO at 100 μM. l-Dehydroascorbate (DHA), an oxidized form of Asc, degraded HO, whereas it did not quench OH or O, which are sources of HO. Furthermore, EGCG efficiently scavenged NTP-induced O, O, and HO in Chelex-treated water. These results indicate that the redox cycling of Asc/DHA and metabolites of DHA are important to be considered when applying NTP to cells and tissues. Additionally, ROS-reducing compounds, such as EGCG, affect the outcome. Further studies are warranted to elucidate the interaction between ROS and biomolecules to promote the medical applications of NTP.
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http://dx.doi.org/10.1016/j.abb.2021.108762DOI Listing
March 2021

Carcinogenesis as Side Effects of Iron and Oxygen Utilization: From the Unveiled Truth toward Ultimate Bioengineering.

Cancers (Basel) 2020 Nov 10;12(11). Epub 2020 Nov 10.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya 466-8550, Japan.

Evolution from the first life on earth to humans took ~3.8 billion years. During the time there have been countless struggles among the species. Mycobacterium tuberculosis was the last major uncontrollable species against the human public health worldwide. After the victory with antibiotics, cancer has become the leading cause of death since 1981 in Japan. Considering that life inevitably depends on ceaseless electron transfers through iron and oxygen, we believe that carcinogenesis is intrinsically unavoidable side effects of using iron and oxygen. Many animal models unequivocally revealed that excess iron is a risk for carcinogenesis. This is supported by a variety of human epidemiological data on cancer risk and prognosis. Cancer is basically a disease of the genome with persistently activated oncogenes and inactivated tumor suppressor genes through which iron addiction with ferroptosis-resistance is maintained. Engineering has made a great advance in the past 50 years. In particular, nanotechnology is distinct in that the size of the engineered molecules is similar to that of our biomolecules. While some nano-molecules are found carcinogenic, there are principles to avoid such carcinogenicity with a smart possibility to use nano-molecules to specifically kill cancer cells. Non-thermal plasma is another modality to fight against cancer.
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http://dx.doi.org/10.3390/cancers12113320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698037PMC
November 2020

Asbestos conceives Fe(II)-dependent mutagenic stromal milieu through ceaseless macrophage ferroptosis and β-catenin induction in mesothelium.

Redox Biol 2020 09 24;36:101616. Epub 2020 Jun 24.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan; Sydney Medical School, The University of Sydney, NSW, 2006, Australia. Electronic address:

Asbestos is still a social burden worldwide as a carcinogen causing malignant mesothelioma. Whereas recent studies suggest that local iron reduction is a preventive strategy against carcinogenesis, little is known regarding the cellular and molecular mechanisms surrounding excess iron. Here by differentially using high-risk and low-risk asbestos fibers (crocidolite and anthophyllite, respectively), we identified asbestos-induced mutagenic milieu for mesothelial cells. Rat and cell experiments revealed that phagocytosis of asbestos by macrophages results in their distinctive necrotic death; initially lysosome-depenent cell death and later ferroptosis, which increase intra- and extra-cellular catalytic Fe(II). DNA damage in mesothelial cells, as assessed by 8-hydroxy-2'-deoxyguanosine and γ-H2AX, increased after crocidolite exposure during regeneration accompanied by β-catenin activation. Conversely, β-catenin overexpression in mesothelial cells induced higher intracellular catalytic Fe(II) with increased G2/M cell-cycle fraction, when p16 genomic loci localized more peripherally in the nucleus. Mesothelial cells after challenge of HO under β-catenin overexpression presented low p16 expression with a high incidence of deletion in p16 locus. Thus, crocidolite generated catalytic Fe(II)-rich mutagenic environment for mesothelial cells by necrotizing macrophages with lysosomal cell death and ferroptosis. These results suggest novel molecular strategies to prevent mesothelial carcinogenesis after asbestos exposure.
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http://dx.doi.org/10.1016/j.redox.2020.101616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330611PMC
September 2020

Overexpression of miR-199/214 is a distinctive feature of iron-induced and asbestos-induced sarcomatoid mesothelioma in rats.

Cancer Sci 2020 Jun 5;111(6):2016-2027. Epub 2020 May 5.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Malignant mesothelioma (MM) is one of the most lethal tumors in humans. The onset of MM is linked to exposure to asbestos, which generates reactive oxygen species (ROS). ROS are believed to be derived from the frustrated phagocytosis and the iron in asbestos. To explore the pathogenesis of MM, peritoneal MM was induced in rats by the repeated intraperitoneal injection of iron saccharate and nitrilotriacetate. In the present study, we used microarray techniques to screen the microRNA (miR) expression profiles of these MM. We observed that the histological subtype impacted the hierarchical clustering of miR expression profiles and determined that miR-199/214 is a distinctive feature of iron saccharate-induced sarcomatoid mesothelioma (SM). Twist1, a transcriptional regulator of the epithelial-mesenchymal transition, has been shown to activate miR-199/214 transcription; thus, the expression level of Twist1 was examined in iron-induced and asbestos-induced mesotheliomas in rats. Twist1 was exclusively expressed in iron saccharate-induced SM but not in the epithelioid subtype. The Twist1-miR-199/214 axis is activated in iron saccharate-induced and asbestos-induced SM. The expression levels of miR-214 and Twist1 were correlated in an asbestos-induced MM cell line, suggesting that the Twist1-miR-199/214 axis is preserved. MeT5A, an immortalized human mesothelial cell line, was used for the functional analysis of miR. The overexpression of miR-199/214 promoted cellular proliferation, mobility and phosphorylation of Akt and ERK in MeT5A cells. These results indicate that miR-199/214 may affect the aggressive biological behavior of SM.
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http://dx.doi.org/10.1111/cas.14405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293088PMC
June 2020

deficiency provides longer survival upon intraperitoneal crocidolite injection in female mice.

Free Radic Res 2020 Mar;54(2-3):195-205

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.

Exposure to asbestos fiber is central to mesothelial carcinogenesis. Recent sequencing studies on human and rodent malignant mesothelioma (MM) revealed frequently mutated genes, including , and . Crocidolite directly or indirectly catalyses the generation of hydroxyl radicals, which appears to be the major driving force for mesothelial mutations. DNA base modification is an oxidative DNA damage mechanism, where 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the most abundant modification both physiologically and pathologically. Multiple distinct mechanisms work together to decrease the genomic level of 8-OHdG through the enzymatic activities of and . Knockout of one or multiple enzymes is not lethal but increases the incidence of tumors. Here, we used single knockout (KO) mice to test whether the deficiency of these three genes affects the incidence and prognosis of asbestos-induced MM. Intraperitoneal injection of 3 mg crocidolite induced MM at a fraction of 14.8% (4/27) in KO, 41.4% (12/29) in KO and 24.0% (6/25) in KO mice, whereas 31.7% (20/63) induction was observed in wild-type (Wt) mice. The lifespan of female KO mice was longer than that of female Wt mice ( = 0.0468). Whole genome scanning of MM with array-based comparative genomic hybridization revealed rare genomic alterations compared to MM in rats and humans. These results indicate that neither Mutyh deficiency nor Ogg1 deficiency promotes crocidolite-induced MM in mice, but the sanitizing nucleotide pool with Mth1 is advantageous in crocidolite-induced mesothelial carcinogenesis.
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http://dx.doi.org/10.1080/10715762.2020.1743285DOI Listing
March 2020

Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats.

Cancer Sci 2020 Apr 21;111(4):1180-1192. Epub 2020 Mar 21.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2-day time-lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array-based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.
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http://dx.doi.org/10.1111/cas.14358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156836PMC
April 2020

Oxidative stress-dependent and -independent death of glioblastoma cells induced by non-thermal plasma-exposed solutions.

Sci Rep 2019 09 20;9(1):13657. Epub 2019 Sep 20.

Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.

Non-thermal atmospheric pressure plasma has been widely used for preclinical studies in areas such as wound healing, blood coagulation, and cancer therapy. We previously developed plasma-activated medium (PAM) and plasma-activated Ringer's lactate solutions (PAL) for cancer treatments. Many in vitro and in vivo experiments demonstrated that both PAM and PAL exhibit anti-tumor effects in several types of cancer cells such as ovarian, gastric, and pancreatic cancer cells as well as glioblastoma cells. However, interestingly, PAM induces more intracellular reactive oxygen species in glioblastoma cells than PAL. To investigate the differences in intracellular molecular mechanisms of the effects of PAM and PAL in glioblastoma cells, we measured gene expression levels of antioxidant genes such as CAT, SOD2, and GPX1. Microarray and quantitative real-time PCR analyses revealed that PAM elevated stress-inducible genes that induce apoptosis such as GADD45α signaling molecules. PAL suppressed genes downstream of the survival and proliferation signaling network such as YAP/TEAD signaling molecules. These data reveal that PAM and PAL induce apoptosis in glioblastoma cells by different intracellular molecular mechanisms.
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http://dx.doi.org/10.1038/s41598-019-50136-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754505PMC
September 2019

l-Dehydroascorbic acid recycled by thiols efficiently scavenges non-thermal plasma-induced hydroxyl radicals.

Arch Biochem Biophys 2019 07 31;669:87-95. Epub 2019 May 31.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan. Electronic address:

Recent development in electronics has enabled the use of non-thermal plasma (NTP) to strictly direct oxidative stress in a defined location at near-physiological temperature. In preclinical studies or human clinical trials, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of free radicals in liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here we quantitated hydroxyl radicals, a major reactive oxygen species generated after NTP exposure, by electron paramagnetic resonance (EPR) spectroscopy using two distinct spin-trapping probes, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), in the presence of thiols or antioxidants. l-Ascorbic acid (AsA) at 25-50 μM concentrations (physiological concentration in the serum) significantly scavenged these hydroxyl radicals, whereas dithiothreitol (DTT), reduced glutathione (GSH), and N-acetyl-cysteine (NAC) as thiols were required in millimolar concentrations to perform scavenging activities. l-Dehydroascorbic acid (DHA), an oxidized form of AsA, necessitated the presence of 25-50 μM DTT or sub-millimolar concentrations of GSH and NAC for the scavenging of hydroxyl radicals and failed to scavenge hydroxyl radicals by itself. These results suggest that the redox cycling of AsA/DHA via thiols and cellular AsA metabolism are important processes to be considered while applying NTP to cells and tissues. Further studies are warranted to elucidate the interaction between other reactive species generated by NTP and biomolecules to promote biological and medical applications of NTP.
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http://dx.doi.org/10.1016/j.abb.2019.05.019DOI Listing
July 2019

Twist1 was detected in mesenchymal cells of mammary fibroadenoma and invasive components of breast carcinoma in rats.

J Toxicol Pathol 2019 Jan 1;32(1):19-26. Epub 2018 Sep 1.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.

Fibroadenoma (FA) is a common mammary fibroepithelial tumor. The tumor size of the FA is increased by estrogen, progesterone, prolactin, and pregnancy, whereas it decreases after menopause. These observations in humans indicate that FA is hormone dependent. In rats, the most common mammary neoplasm is also FA. Expression levels of Twist1, a transcriptional regulator of epithelial-mesenchymal transition, were examined in paraffin-embedded tissue sections of an experimental rat breast model to find physiological alternations coincident with reproductive hormonal changes. Twenty-three Fischer 344/Brown Norway F1 hybrid rats were used as 14- to 16-week-old adolescent rats (n=3), pregnant rats (n=4), and lactating rats (n=6) in addition to rats over 100-weeks-old that exhibited aging (n=3) and FA (n=7). Seventy-six cases of chemically induced breast carcinoma and two cases of FA in Sprague Dawley rats were also examined. Using tissue sections, we observed that Twist1-positive mesenchymal cells were predominantly located in the periductal region in adolescent and pregnant rats and in the terminal duct lobular unit in pregnant and elderly rats. Twist1 was also expressed diffusely in the mesenchymal cells of FA rats. Twist1-positive cancer-associated mesenchymal cells were found more frequently in the invasive components of breast carcinomas than in intraductal components. The expressions of Twist1 in mesenchymal cells were induced by physiological and pathological stimuli, suggesting the biological role of Twist1 in tissue structure. Further study may reveal the role of Twist1 in mesenchymal cells of mammary glands in rats.
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http://dx.doi.org/10.1293/tox.2018-0029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361665PMC
January 2019

Global overexpression of divalent metal transporter 1 delays crocidolite-induced mesothelial carcinogenesis in male mice.

Free Radic Res 2018 Sep 12;52(9):1030-1039. Epub 2018 Oct 12.

a Department of Pathology and Biological Responses , Nagoya University Graduate School of Medicine , Nagoya , Japan.

Exposure to asbestos fiber is central to mesothelial carcinogenesis, for which iron overload in or near mesothelial cells is a key pathogenic mechanism. Alternatively, iron chelation therapy with deferasirox or regular phlebotomy was significantly preventive against crocidolite-induced mesothelial carcinogenesis in rats. However, the role of iron transporters during asbestos-induced carcinogenesis remains elusive. Here, we studied the role of divalent metal transporter 1 (DMT1; Slc11a2), which is a Fe(II) transporter, that is present not only on the apical plasma membrane of duodenal cells but also on the lysosomal membrane of every cell, in crocidolite-induced mesothelial carcinogenesis using DMT1 transgenic (DMT1Tg) mice. DMT1Tg mice show mucosal block of iron absorption without cancer susceptibility under normal diet. We unexpectedly found that superoxide production was significantly decreased upon stimulation with crocidolite both in neutrophils and macrophages of DMT1Tg mice, and the macrophage surface revealed higher iron content 1 h after contact with crocidolite. Intraperitoneal injection of 3 mg crocidolite ultimately induced malignant mesothelioma in ∼50% of both wild-type and DMT1Tg mice (23/47 and 14/28, respectively); this effect was marginally (p = 0.069) delayed in DMT1Tg mice, promoting survival. The promotional effect of nitrilotriacetic acid was limited, and the liver showed significantly higher iron content both in DMT1Tg mice and after crocidolite exposure. The results indicate that global DMT1 overexpression causes decreased superoxide generation upon stimulation in inflammatory cells, which presumably delayed the promotional stage of crocidolite-induced mesothelial carcinogenesis. DMT1Tg mice with low-stamina inflammatory cells may be helpful to evaluate the involvement of inflammation in various pathologies.
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http://dx.doi.org/10.1080/10715762.2018.1514604DOI Listing
September 2018

A fluorescent metal-sensor study provides evidence for iron transport by transcytosis in the intestinal epithelial cells.

J Clin Biochem Nutr 2018 Jan 12;62(1):49-55. Epub 2017 Dec 12.

Feist-Weiller Cancer Center and the Department of Medicine, Louisiana State University Health Sciences Center-Shreveport, 1501 Kings Highway, Shreveport, LA 71130, USA.

Iron transport across the intestinal epithelium is facilitated by the divalent metal transporter 1 (DMT1) on the brush border membrane (BBM). The fluorescent metal sensor calcein, which is hydrophilic, membrane-impermeable and quenched by chelation with iron, was used to test our hypothesis that intestinal iron absorption is through the endocytic processes and is involved in a pathway where BBM-derived vesicles fuse with basolateral membrane (BLM)-derived vesicles. To monitor the flux of iron via transcytosis, Caco-2 cells were employed as a polarized cell layer in Transwell chambers. When calcein was added to the basal chamber along with apo-transferrin (apo-Tf), calcein rapidly underwent endocytosis and co-localized with apo-Tf. Calcein was quenched by adding an iron-ascorbate complex and then restored by adding 2,2'-dipyridyl into the apical chamber. These results were confirmed by live-cell imaging. When hemin from the apical surface and calcein from the basal chamber were added to the Caco-2 cells, internalization of DMT1 and quenching of calcein were not observed until 2 h later. These results indicated that absorbed hemin required processing before hemin-derived iron was available to BLM-derived vesicles. These studies suggest that iron is transported in Caco-2 cells by transcytosis with apical-derived vesicles that are fused to BLM-derived vesicles.
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http://dx.doi.org/10.3164/jcbn.17-74DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773836PMC
January 2018

Role of catalytic iron and oxidative stress in nitrofen-induced congenital diaphragmatic hernia and its amelioration by Saireito (TJ-114).

J Clin Biochem Nutr 2017 Nov 5;61(3):176-182. Epub 2017 Sep 5.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Congenital diaphragmatic hernia (CDH) is a life-threatening neonatal disease that leads to lung hypoplasia and pulmonary hypertension. We recently found that maternal prenatal administration of Saireito (TJ-114) ameliorates fetal CDH in a nitrofen-induced rat model. Here, we studied the role of iron and oxidative stress in neonates of this model and in lung fibroblasts IMR90-SV in association with nitrofen and Saireito. We observed increased immunostaining of 8-hydroxy-2'-deoxyguanosine in the lungs of neonates with CDH, which was ameliorated by maternal Saireito intake. Pulmonary expression was significantly decreased in both CDH and CDH after Saireito in comparison to normal controls, indicating functional lung immaturity, whereas catalytic Fe(II) and pulmonary expression remained constant among the three groups. Saireito revealed a dose-dependent scavenging capacity with electron spin resonance spin trapping against hydroxyl radicals but not against superoxide. Finally, nitrofen revealed dose-dependent cytotoxicity to IMR90-SV cells, accompanied by an increase in oxidative stress, as seen by 5(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and catalytic Fe(II). Saireito ameliorated all of these in IMR90-SV cells. In conclusion, catalytic Fe(II)-dependent oxidative stress by nitrofen may be the pathogenic cause of CDH, and the antioxidative activity of Saireito is at least partially responsible for improving nitrofen-induced CDH.
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http://dx.doi.org/10.3164/jcbn.17-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703780PMC
November 2017

Phlebotomy as a preventive measure for crocidolite-induced mesothelioma in male rats.

Cancer Sci 2018 Feb 4;109(2):330-339. Epub 2018 Jan 4.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Malignant mesothelioma (MM) is a rare but socially important neoplasm due to its association with asbestos exposure. Malignant mesothelioma is difficult to diagnose at an early stage, yet there are no particularly effective treatments available at the advanced stage, thus necessitating efficient strategies to prevent MM in individuals already exposed to asbestos. We previously showed that persistent oxidative damage caused by foreign body reaction and affinity of asbestos both to hemoglobin and histones is one of the major pathogeneses. Accordingly, as an effective strategy to prevent asbestos-induced MM, we undertook the use of an iron chelator, deferasirox, which decreased the epithelial-mesenchymal transition in a crocidolite-induced rat MM model. However, this agent may show adverse effects. Here, we studied the effects of iron removal by phlebotomy as a realistic measure on the same rat model. We injected a total of 5 mg crocidolite i.p. to F1 hybrid rats between the Fischer-344 and Brown-Norway strains at the age of 6 weeks. We repeated weekly or biweekly phlebotomy of 6-8 mL/kg/time from 10 to 60 weeks of age. The animals were observed until 120 weeks. In male rats, phlebotomy significantly decreased the weight and nuclear grade of MM, and modestly reduced the associated ascites and the fraction of more malignant sarcomatoid subtype. Weekly phlebotomy prolonged long-term survival. Our results indicate that appropriate phlebotomy may be a practical preventive measure to attenuate the initiation and promotion capacity of asbestos towards MM by reducing iron in individuals exposed to asbestos.
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http://dx.doi.org/10.1111/cas.13460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797813PMC
February 2018

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells.

Biochem Biophys Rep 2017 Jul 27;10:26-31. Epub 2017 Feb 27.

Feist-Weiller Cancer Center and the Department of Medicine, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA 71130, USA.

Background: Protoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.

Methods: We have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses.

Results: PP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.

Conclusion: We hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.

General Significance: These results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.
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http://dx.doi.org/10.1016/j.bbrep.2017.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614651PMC
July 2017

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats.

J Clin Biochem Nutr 2017 Jul 15;61(1):18-24. Epub 2017 Jun 15.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.

Daily intake of vegetables can reduce the risk of cancer and lifestyle-related diseases. However, supplementary intake of β-carotene alone has been reported to increase the risk of lung cancer in male cigarette smokers and people who were exposed to asbestos. The mechanism of the antioxidative properties of carotenoids , especially under oxidative stress conditions, still remains unclear. To investigate the antioxidant properties of dietary compounds, we examined the effects of chemically modified astaxanthin (Ax-C-8) using a rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative injury. Ax-C-8 demonstrated lethally toxic effects on the rats in a dose-dependent manner. Following supplementation with Ax-C-8 (0.02%, w/w) for 30 days, the rats were euthanized 1, 4 and 24 h after injection of Fe-NTA. After 4 h, Ax-C-8 pretreatment suppressed the elevation of creatinine and blood urea nitrogen and protected the rats from renal tubular necrosis and the formation of 4-hydroxy-2-nonenal-modified proteins. After 24 h, pretreatment with Ax-C-8 maintained the renal antioxidant enzyme levels and renal tubules. Here, we demonstrate the antioxidant effects of Ax-C-8 against Fe-NTA-induced oxidative injury in rats receiving a regular diet. These data suggest that dietary intake of astaxanthin may be useful for the prevention of renal tubular oxidative damage.
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http://dx.doi.org/10.3164/jcbn.16-114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5525010PMC
July 2017

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics.

Sci Rep 2017 05 12;7(1):1821. Epub 2017 May 12.

Division of Genome Medicine, Institute for Genome Research, Tokushima University, Tokushima, 770-8503, Japan.

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.
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http://dx.doi.org/10.1038/s41598-017-01951-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431889PMC
May 2017

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy.

Free Radic Biol Med 2017 07 2;108:904-917. Epub 2017 May 2.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address:

Non-thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti-proliferative efficacy. Interestingly, NTP demonstrated greater selective anti-proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti-proliferative effect of NTP was enhanced by pre-incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM-H2DCFDA, MitoSOX and C11-BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor-1 and increased ferritin-H and -L chain expression that was correlated with decreased iron-regulatory protein expression and RNA-binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)-selective probe, HMRhoNox-M, and was consistent with autophagic-induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid-phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B-II expression, which co-localized with LAMP1, indicating autophagolysosome formation. Autophagic-induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid-phase endocytosis, lysosome biogenesis and autophagy.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.368DOI Listing
July 2017

Iron and thiol redox signaling in cancer: An exquisite balance to escape ferroptosis.

Free Radic Biol Med 2017 07 19;108:610-626. Epub 2017 Apr 19.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Epidemiological data indicate a constant worldwide increase in cancer mortality, although the age of onset is increasing. Recent accumulation of genomic data on human cancer via next-generation sequencing confirmed that cancer is a disease of genome alteration. In many cancers, the Nrf2 transcription system is activated via mutations either in Nrf2 or Keap1 ubiquitin ligase, leading to persistent activation of the genes with antioxidative functions. Furthermore, deep sequencing of passenger mutations is clarifying responsible cancer causative agent(s) in each case, including aging, APOBEC activation, smoking and UV. Therefore, it is most likely that oxidative stress is the principal initiating factor in carcinogenesis, with the involvement of two essential molecules for life, iron and oxygen. There is evidence based on epidemiological and animal studies that excess iron is a major risk for carcinogenesis, suggesting the importance of ferroptosis-resistance. Microscopic visualization of catalytic Fe(II) has recently become available. Although catalytic Fe(II) is largely present in lysosomes, proliferating cells harbor catalytic Fe(II) also in the cytosol and mitochondria. Oxidative stress catalyzed by Fe(II) is counteracted by thiol systems at different functional levels. Nitric oxide, carbon monoxide and hydrogen (per)sulfide modulate these reactions. Mitochondria generate not only energy but also heme/iron sulfur cluster cofactors and remain mostly dysfunctional in cancer cells, leading to Warburg effects. Cancer cells are under persistent oxidative stress with a delicate balance between catalytic iron and thiols, thereby escaping ferroptosis. Thus, high-dose L-ascorbate and non-thermal plasma as well as glucose/glutamine deprivation may provide additional benefits as cancer therapies over preexisting therapeutics.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.024DOI Listing
July 2017

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma.

Free Radic Biol Med 2017 05 6;106:91-99. Epub 2017 Feb 6.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address:

CD44 exists as a standard (CD44s) isoform and different variant isoforms (CD44v) due to alternative splicing. While the complex nature of these different isoforms has not been fully elucidated, CD44v expression has been shown to exert oncogenic effects by promoting tumor progression, metastasis and resistance of tumor cells to chemotherapy. One of the CD44v isoforms, CD44v8-10, was recently shown to protect cancer cells from oxidative stress by increasing the synthesis of glutathione (GSH). However, data regarding CD44 isoform expression in malignant mesothelioma (MM) are still lacking. Here, we show that most of the MM cell lines express both the CD44s and CD44v isoforms, in contrast to non-tumorigenic mesothelial cells, which express only CD44s. Moreover, we show here that these MM cell lines are positive for CD44 variable exon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable exon 9 was found to be statistically associated with NF2 inactivation, a common occurrence in MM. Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. Our results thus suggest that the response to CD44 depletion is cell type-dependent and, in cases such as MM cells, compensatory pathway(s) might be activated rheostatically to account for the loss of CD44 and counteract enhanced oxidative stress.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.011DOI Listing
May 2017

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor.

Cancer Sci 2017 Apr 20;108(4):785-794. Epub 2017 Apr 20.

Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan.

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.
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http://dx.doi.org/10.1111/cas.13175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406609PMC
April 2017

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells.

Arch Biochem Biophys 2016 09 24;605:109-16. Epub 2016 May 24.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address:

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by l-ascorbate. l-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM l-ascorbate. However, brief pre-treatment with a pharmacological dose (250-750 μM) of l-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of l-ascorbate was completely abolished by a prolonged 4 h pre-incubation with l-ascorbate (500 μM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2',7'-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and l-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.
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http://dx.doi.org/10.1016/j.abb.2016.05.016DOI Listing
September 2016

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis.

Cancer Sci 2016 Mar 18;107(3):250-7. Epub 2016 Feb 18.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Multi-wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50-nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.
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http://dx.doi.org/10.1111/cas.12865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814253PMC
March 2016

Asbestos and multi-walled carbon nanotubes generate distinct oxidative responses in inflammatory cells.

J Clin Biochem Nutr 2015 Mar 28;56(2):111-7. Epub 2014 Nov 28.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Asbestos exposure is considered a social burden by causing mesothelioma. Despite the use of synthetic materials, multi-walled carbon nanotubes (MWCNTs) are similar in dimension to asbestos and produce mesothelioma in animals. The role of inflammatory cells in mesothelial carcinogenesis remains unclear. Here, we evaluated the differences in inflammatory cell responses following exposure to these fibrous materials using a luminometer and L-012 (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione) to detect reactive oxygen species (ROS). Rat peripheral blood or RAW264.7 cells were used to assess the effects on neutrophils and macrophages, respectively. Crocidolite and amosite induced significant ROS generation by neutrophils with a peak at 10 min, whereas that of chrysotile was ~25% of the crocidolite/amosite response. MWCNTs with different diameters (~15, 50, 115 and 145 nm) and different carcinogenicity did not induce significant ROS in peripheral blood. However, the MWCNTs induced a comparable amount of ROS in RAW264.7 cells to that following asbestos treatment. The peaks for MWCNTs (0.5-1.5 h) were observed earlier than those for asbestos (1-5 h). Apocynin and superoxide dismutase significantly inhibited ROS generation for each fiber, suggesting an involvement of NADPH oxidase and superoxide. Thus, asbestos and MWCNTs induce different oxidative responses in inflammatory cells, indicating the importance of mesothelial cell evaluation for carcinogenesis.
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http://dx.doi.org/10.3164/jcbn.14-92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345184PMC
March 2015

Direct exposure of non-equilibrium atmospheric pressure plasma confers simultaneous oxidative and ultraviolet modifications in biomolecules.

J Clin Biochem Nutr 2014 Nov 9;55(3):207-15. Epub 2014 Sep 9.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Thermal plasmas and lasers are used in medicine to cut and ablate tissues and for coagulation. Non-equilibrium atmospheric pressure plasma (NEAPP) is a recently developed, non-thermal technique with possible biomedical applications. Although NEAPP reportedly generates reactive oxygen/nitrogen species, electrons, positive ions, and ultraviolet radiation, little research has been done into the use of this technique for conventional free radical biology. Recently, we developed a NEAPP device with high electron density. Electron spin resonance spin-trapping revealed (•)OH as a major product. To obtain evidence of NEAPP-induced oxidative modifications in biomolecules and standardize them, we evaluated lipid peroxidation and DNA modifications in various in vitro and ex vivo experiments. Conjugated dienes increased after exposure to linoleic and α-linolenic acids. An increase in 2-thiobarbituric acid-reactive substances was also observed after exposure to phosphatidylcholine, liposomes or liver homogenate. Direct exposure to rat liver in saline produced immunohistochemical evidence of 4-hydroxy-2-nonenal- and acrolein-modified proteins. Exposure to plasmid DNA induced dose-dependent single/double strand breaks and increased the amounts of 8-hydroxy-2'-deoxyguanosine and cyclobutane pyrimidine dimers. These results indicate that oxidative biomolecular damage by NEAPP is dose-dependent and thus can be controlled in a site-specific manner. Simultaneous oxidative and UV-specific DNA damage may be useful in cancer treatment.
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http://dx.doi.org/10.3164/jcbn.14-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227828PMC
November 2014

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis.

Nagoya J Med Sci 2014 Feb;76(1-2):149-60

Asbestos was abundantly used in industry during the last century. Currently, asbestos confers a heavy social burden due to an increasing number of patients with malignant mesothelioma (MM), which develops after a long incubation period. Many studies have been conducted on the effects of the asbestos types that were most commonly used for commercial applications. However, there are few studies describing the effects of the less common types, or minor asbestos. We performed a rat carcinogenesis study using Japanese tremolite and Afghan anthophyllite. Whereas more than 50% of tremolite fibers had a diameter of < 500 nm, only a small fraction of anthophyllite fibers had a diameter of < 500 nm. We intraperitoneally injected 1 or 10 mg of asbestos into F1 Fischer-344/Brown-Norway rats. In half of the animals, repeated intraperitoneal injections of nitrilotriacetate (NTA), an iron chelator to promote Fenton reaction, were performed to evaluate the potential involvement of iron overload. Tremolite induced MM with a high incidence (96% with 10 mg; 52% with 1 mg), and males were more susceptible than females. Histology was confirmed using immunohistochemistry, and most MMs were characterized as the sarcomatoid or biphasic subtype. Unexpectedly NTA showed an inhibitory effect in females. In contrast, anthophyllite induced no MM after an observation period of 550 days. The results suggest that the carcinogenicity of anthophyllite is weaker than formerly reported, whereas that of tremolite is as potent as major asbestos as compared with our previous data.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345736PMC
February 2014

Connective tissue growth factor and β-catenin constitute an autocrine loop for activation in rat sarcomatoid mesothelioma.

J Pathol 2014 Aug 16;233(4):402-14. Epub 2014 Jun 16.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Japan.

Due to the formerly widespread use of asbestos, malignant mesothelioma (MM) is increasingly frequent worldwide. MM is classified into epithelioid (EM), sarcomatoid (SM), and biphasic subtypes. SM is less common than EM but is recognized as the most aggressive type of MM, and these patients have a poor prognosis. To identify genes responsible for the aggressiveness of SM, we induced EM and SM in rats, using asbestos, and compared their transcriptomes. Based on the results, we focused on connective tissue growth factor (Ctgf), whose expression was significantly increased in SM compared with EM; EM itself exhibited an increased expression of Ctgf compared with normal mesothelium. Particularly in SM, Ctgf was a major regulator of MM proliferation and invasion through activation of the β-catenin-TCF-LEF signalling pathway, which is autocrine and formed a positive feedback loop via LRP6 as a receptor for secreted Ctgf. High Ctgf expression also played a role in the epithelial-mesenchymal transition in MM. Furthermore, Ctgf is a novel serum biomarker for both early diagnosis and determining the MM prognosis in rats. These data link Ctgf to SM through the LRP6-GSK3β-β-catenin-TCF-Ctgf autocrine axis and suggest Ctgf as a therapeutic target.
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http://dx.doi.org/10.1002/path.4377DOI Listing
August 2014

Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats.

Pathol Int 2013 Sep;63(9):457-62

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine.

Multiwalled carbon nanotubes (MWCNTs) have attracted public attention not only for their potential applications in engineering and materials science but also for possible environmental risks. MWCNTs share similar properties with asbestos, a definite human carcinogen causing malignant mesothelioma (MM), in that they are both biopersistent thin fibers with a high aspect ratio. Certain types of MWCNTs do induce MM in animal experiments. Though there are many different types of MWCNTs awaiting use in industry, there is little evidence about what types of MWCNTs present a high risk for MM in vivo. We have previously shown that the diameter of MWCNTs is one of the critical factors for mesothelial injury, which eventually leads to MM. Because of the extensive commercial use of MWCNTs, the properties of MWCNTs that determine carcinogenic activity should be clarified. Here we report that a high dose (10 mg) of a tangled form of pristine MWCNT (with a diameter of 15 nm) did not induce MM after intraperitoneal administration in rats, which were followed for up to 3 years after injection. This observation strengthens our previous finding that the rigidity, diameter, length and surface properties of MWCNTs are important factors in MM induction in vivo.
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http://dx.doi.org/10.1111/pin.12093DOI Listing
September 2013

Deferasirox induces mesenchymal-epithelial transition in crocidolite-induced mesothelial carcinogenesis in rats.

Cancer Prev Res (Phila) 2013 Nov 11;6(11):1222-30. Epub 2013 Sep 11.

Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.

Asbestos was used worldwide in huge quantities in the past century. However, because of the unexpected carcinogenicity to mesothelial cells with an extremely long incubation period, many countries face this long-lasting social problem. Mesothelioma is often diagnosed in an advanced stage, for which no effective therapeutic protocols are yet established. We previously reported on the basis of animal experiments that the major pathology in asbestos-induced mesothelial carcinogenesis is local iron overload. Here, we undertook to find an effective strategy to prevent, delay, or lower the malignant potential of mesothelioma during asbestos-induced carcinogenesis. We used intraperitoneal injections of crocidolite to rats. We carried out a 16-week study to seek the maximal-tolerated intervention for iron reduction via oral deferasirox administration or intensive phlebotomy. Splenic iron deposition was significantly decreased with either method, and we found that Perls' iron staining in spleen is a good indicator for iron reduction. We injected a total of 10 mg crocidolite at the age of six weeks, and the preventive measures were via repeated oral administration of 25 to 50 mg/kg/d deferasirox or weekly to bimonthly phlebotomy of 4 to 10 mL/kg/d. The animals were observed until 110 weeks. Deferasirox administration significantly increased the fraction of less malignant epithelioid subtype. Although we found a slightly prolonged survival in deferasirox-treated female rats, larger sample size and refinement of the current protocol are necessary to deduce the cancer-preventive effects of deferasirox. Still, our results suggest deferasirox serves as a potential preventive strategy in people already exposed to asbestos via iron reduction.
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http://dx.doi.org/10.1158/1940-6207.CAPR-13-0244DOI Listing
November 2013