Tatsuya Kato

Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo assay systems

Abstract The genotoxic effects of multi-walled carbon nanotubes (MWCNTs) were examined by using in vitro and in vivo assays. MWCNTs significantly induced micronuclei in A549 cells and enhanced the frequency of sister chromatid exchange (SCE) in CHO AA8 cells. When ICR mice were intratracheally instilled with a single dose (0.05 or 0.2 mg/animal) of MWCNTs, DNA damage of the lungs, analysed by comet assay, increased in a dose-dependent manner. Moreover, DNA oxidative damage, indicated by 8-oxo-7,8-dihydro-2′-deoxyguanosine and heptanone etheno-deoxyribonucleosides, occurred in the lungs of MWCNT-exposed mice. The gpt mutation frequencies significantly increased in the lungs of MWCNT-treated gpt delta transgenic mice. Transversions were predominant, and G:C to C:G was clearly increased by MWCNTs. Moreover, many regions immunohistochemically stained for inducible NO synthase and nitrotyrosine were observed in the lungs of MWCNT-exposed mice. Overall, MWCNTs were shown to be genotoxic both in in vitro and in vivo tests; the mechanisms probably involve oxidative stress and inflammatory responses.

Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

BackgroundRecently, manufactured nano/microparticles such as fullerenes (C60), carbon black (CB) and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN) test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles.ResultsIn in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations.ConclusionManufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

Endoscopic indocyanine green video angiography in aneurysm surgery: an innovative method for intraoperative assessment of blood flow in vasculature hidden from microscopic view

Recently, intraoperative fluorescence video angiography using indocyanine green (ICG) has been widely used in aneurysm surgery. This is a simple and useful method to confirm complete occlusion of the aneurysm lumen and preservation of blood flow in the arteries around the aneurysm. However, the observation field of ICG video angiography is limited under a microscope, making it difficult to confirm the flow in the arteries behind the parent arteries or aneurysm. The authors developed a new technique of intraoperative endoscopic ICG video angiography to assess the blood flow in perforating arteries hidden by the parent arteries or aneurysm. The endoscope emits excitation light with a wavelength of approximately 800 nm, and video images were obtained through a cut filter. The authors used this ICG fluorescence endoscope in treating 3 patients with unruptured cerebral aneurysms. During clip placement, the endoscope was inserted to confirm aneurysm occlusion. Then, ICG was intravenously administered, and the fluorescence in the vessels was observed via the endoscope as well as under the microscope. The blood flow in the perforating arteries was clearly identified, and no procedural complication occurred. The authors conclude that the technique is very useful and facilitates intraoperative real-time assessment of the patency of perforating arteries behind parent arteries or aneurysms.

Magnetite Nanoparticles Induce Genotoxicity in the Lungs of Mice via Inflammatory Response

Nanomaterials are useful for their characteristic properties and are commonly used in various fields. Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields, whereas their toxicological properties are not well documented. A safety assessment is thus urgently required for MGT, and genotoxicity is one of the most serious concerns. In the present study, we examined genotoxic effects of MGT using mice and revealed that DNA damage analyzed by a comet assay in the lungs of imprinting control region (ICR) mice intratracheally instilled with a single dose of 0.05 or 0.2 mg/animal of MGT was approximately two- to three-fold higher than that of vehicle-control animals. Furthermore, in gpt delta transgenic mice, gpt mutant frequency (MF) in the lungs of the group exposed to four consecutive doses of 0.2 mg MGT was significantly higher than in the control group. Mutation spectrum analysis showed that base substitutions were predominantly induced by MGT, among which G:C to A:T transition and G:C to T:A transversion were the most significant. To clarify the mechanism of mutation caused by MGT, we analyzed the formation of DNA adducts in the lungs of mice exposed to MGT. DNA was extracted from lungs of mice 3, 24, 72 and 168 h after intratracheal instillation of 0.2 mg/body of MGT, and digested enzymatically. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and lipid peroxide-related DNA adducts were quantified by stable isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS). Compared with vehicle control, these DNA adduct levels were significantly increased in the MGT-treated mice. In addition to oxidative stress- and inflammation related-DNA adduct formations, inflammatory cell infiltration and focal granulomatous formations were also observed in the lungs of MGT-treated mice. Based on these findings, it is suggested that inflammatory responses are probably involved in the genotoxicity induced by MGT in the lungs of mice.

Comprehensive DNA Adduct Analysis Reveals Pulmonary Inflammatory Response Contributes to Genotoxic Action of Magnetite Nanoparticles

Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields; however, its toxicological properties are not well documented. In our previous report, MGT showed genotoxicity in both in vitro and in vivo assay systems, and it was suggested that inflammatory responses exist behind the genotoxicity. To further clarify mechanisms underlying the genotoxicity, a comprehensive DNA adduct (DNA adductome) analysis was conducted using DNA samples derived from the lungs of mice exposed to MGT. In total, 30 and 42 types of DNA adducts were detected in the vehicle control and MGT-treated groups, respectively. Principal component analysis (PCA) against a subset of DNA adducts was applied and several adducts, which are deduced to be formed by inflammation or oxidative stress, as the case of etheno-deoxycytidine (εdC), revealed higher contributions to MGT exposure. By quantitative-LC-MS/MS analysis, εdC levels were significantly higher in MGT-treated mice than those of the vehicle control. Taken together with our previous data, it is suggested that inflammatory responses might be involved in the genotoxicity induced by MGT in the lungs of mice.

Effects of valsartan on neuroprotection and neurogenesis after ischemia.

Experimental and clinical studies have revealed that angiotensin II type 1 receptor blocker has protective effects against ischemic brain injury, but the mechanism is still obscure. Angiotensin II type 1 receptor blocker may also have effects on neurogenesis through the activation of unblocked angiotensin II type 2 receptors. In this study, we showed that valsartan significantly suppressed superoxide production and cytochrome C release into the cytosol after transient forebrain ischemia and consequently attenuated ischemic neuronal damage without affecting the blood pressure in rats. However, valsartan has none of the expected effects on neurogenesis after ischemia. These results suggest that valsartan has neuroprotective effects on ischemic injury through the suppression of oxidative stress and mitochondrial injury.

Effect of physicochemical character differences on the genotoxic potency of kaolin

BackgroundKaolin is white clay mineral with the chemical composition Al2Si2O5(OH)4, and many varieties of kaolins having different crystal structures are utilized in industrial, cosmetic and medical fields. To evaluate the effect of physicochemical character differences on the genotoxicity of kaolin, two types of kaolin, kaolin-S with smooth, sphere-shaped crystals, and kaolin-P with clusters of thin pseudohexagonal plates, were used in the study.ResultsICR mice were intratracheally instilled with the kaolins (0.05 and 0.2xa0mg/mouse), and comet assay was performed on their lungs. Both kaolins showed DNA damage in the lungs of the mice, however the DNA damaging potency was much higher with kaolin-P than that with kaolin-S.In order to clarify the mechanisms for the different genotoxic potency, we examined the incorporation rate and ROS generation of these two types of kaolin in alveolar epithelial A549 and macrophage-like RAW264 cells, using flow cytometric (FCM) analysis. Kaolin-P showed a higher incorporation rate into the mammalian cells and ROS generation than that of kaolin-S. Especially, RAW264 cells aggressively incorporated kaolins, and generated ROS, whereas almost no ROS generation was observed in A549 cells. In addition, inflammatory cytokines were quantified, using the ELISA method, to understand further genotoxic potency differences of kaolins. Concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the media were increased by exposure to both kaolins, but in the case of kaolin-P, these inflammatory cytokines were significantly elevated. Based on these findings, differences of genotoxic potency may contribute to incorporation rates into immune cells. Furthermore, it is likely that immune cells and epithelial cells might closely interact with each other for the appearance of genotoxocity in vivo. In order to clarify the interaction between epithelial and immune cells, A549 and RAW264 were co-cultured and RAW264 cells only were exposed to kaolins, then subsequently A549 was applied to FCM analysis and comet assay. DNA damage observed in the A549 cells markedly increased in the presence of kaolin-exposed RAW264 cells compared to the single culture.ConclusionFrom these observations, it is suggested that mechanisms of kaolin genotoxicity against epithelial cells are through the activation of macrophage cells. Therefore, it is thought that interactions between epithelial and immune cells would be very important for evaluation of the genotoxicity of fine particulate matter. We also showed here that co-culture models of epithelial and immune cells could be used as suitable models for evaluation of lung genotoxicity of fine particulate matter, including nanomaterials, as in vivo mimicking systems.

In vivo examination of the genotoxicity of the urban air and surface soil pollutant, 3,6-dinitrobenzo[e]pyrene, with intraperitoneal and intratracheal administration

3,6‐Dinitrobenzo[e]pyrene (3,6‐DNBeP) was identified as a new potent mutagen toward Salmonella strains in surface soil and airborne particles. Because data of in vivo examination of the genotoxicity of 3,6‐DNBeP are limited, micronucleus test was performed in peripheral blood and bone marrow, and comet assay in the lungs of mice treated with 3,6‐DNBeP. In male ICR mice intraperitoneally (i.p.) injected with 3,6‐DNBeP, the frequency of micronuclated polychromatic erythrocytes (MNPCEs) was increased in the peripheral blood and bone marrow after 24 h in a dose‐dependent manner. Compared to controls, the highest dose of 3,6‐DNBeP (40 mg/kg B.W.) induced 7.3‐ and 8.7‐fold increases of MNPCE frequency in the peripheral blood and bone marrow, respectively. Furthermore, when 3,6‐DNBeP was intratracheally (i.t.) instilled to male ICR mice, 3,6‐DNBeP at the highest dose of 0.1 mg/kg body exhibited 3.1‐fold increase of DNA tail moment in the lungs at 3 h after the instillation compared to controls. The values of DNA tail moment at 9 and 24 h after the instillation were increased up to 3.5 and 4.2‐fold, respectively. These data indicate that 3,6‐DNBeP is genotoxic to mammalians in in vivo and suggest that 3,6‐DNBeP may be a carcinogenic compound present in the human environment.

Change in Mutagenic Activity of Genistein after a Nitrite Treatment

This study examined the mutagenic activity of genistein after a nitrite treatment under acidic conditions. Nitrite-treated genistein exhibited mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 with or without S9 mix. Nitrite-treated genistein was demonstrated by electron spin resonance to generate radicals. An instrumental analysis showed 3-nitro-genistein to have been formed in the reaction mixture. However, 3-nitro-genistein did not exhibit mutagenic activity toward the S. typhimurium strains, suggesting that other mutagens might also have been formed in the reaction mixture. The clastogenic properties of nitrite-treated genistein and 3-nitro-genistein were examined by a micronucleus test with male ICR mice. Nitrite-treated genistein and 3-nitro-genistein showed a significantly higher frequency of micronucleated reticulocytes in mice than in the control group. These results suggest that a daily oral intake of genistein and nitrite through foodstuffs might induce the formation of various mutagenic compounds in the body.