Kyoko Hiyoshi

Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

The lung is an organ that is sensitive to mutations induced by chemicals in ambient air, and transgenic mice harboring guanine phosphoribosyltransferase (gpt) gene as a target gene are a well-established model system for assessing genotoxicity in vivo. Transcription factor Nrf2 mediates inducible and constitutive expression of cytoprotective enzymes against xenobiotics and mutagens. To address whether Nrf2 is also involved in DNA protection, we generated nrf2+/-::gpt and nrf2-/-::gpt mice. The spontaneous mutation frequency of the gpt gene in the lung was approximately three times higher in nrf2-null (nrf2-/-) mice than nrf2 heterozygous (nrf2+/-) and wild-type (nrf2+/+) mice, whereas in the liver, the mutation frequency was higher in nrf2-/- and nrf2+/- mice than in nrf2+/+ wild-type mice. By contrast, no difference in mutation frequency was observed in testis among the three genotypes. A single intratracheal instillation of benzo(a)pyrene (BaP) increased the lung mutation frequency 3.1- and 6.1-fold in nrf2+/- and nrf2-/- mice, respectively, compared with BaP-untreated nrf2+/- mice, showing that nrf2-/- mice are more susceptible to genotoxic carcinogens. Surprisingly, mutation profiles of the gpt gene in BaP-treated nrf2+/- mice was substantially different from that in BaP-untreated nrf2-/- mice. In nrf2-/- mice, spontaneous and BaP-induced mutation hotspots were observed at nucleotides 64 and 140 of gpt, respectively. These results thus show that Nrf2 aids in the prevention of mutations in vivo and suggest that Nrf2 protects genomic DNA against certain types of mutations.

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.

Pulmonary toxicity induced by intratracheal instillation of Asian yellow dust (Kosa) in mice

Asian yellow dust (Kosa) causes adverse respiratory health effects in humans. The objective of this study was to clarify the lung toxicity of Kosa. ICR mice (5 weeks of age) were administered intratracheally with Kosa samples-two samples from Maowusu desert and Shapotou desert, one sample consisted of Shapotou Kosa plus sulfate, and natural Asian dust (NAD) from the atmosphere of Beijing-at doses of 0.05, 0.10 or 0.20mg/mouse at four weekly intervals. The four Kosa samples tested had similar compositions of minerals and concentrations of elements. Instillation of dust particles caused bronchitis and alveolitis in treated mice. The magnitude of inflammation was much greater in NAD-treated mice than in the other particles tested. Increased neutrophils, lymphocytes or eosinophils in bronchoalveolar lavage fluids (BALF) of treated mice were dose dependent. The number of neutrophils in BALF at the 0.2mg level was parallel to the content of β-glucan in each particle. The numbers of lymphocytes and eosinophils in BALF at the 0.2mg level were parallel to the concentration of SO(4)(2-) in each particle. Pro-inflammatory mediators-such as interleukin (IL)-12, tumor necrosis factor-(TNF)-α, keratinocyte chemoattractant (KC), monocyte chemotactic protein (MCP)-l and macrophage inflammatory protein-(MIP)-lα in BALF-were greater in the treated mice. Specifically, NAD considerably increased pro-inflammatory mediators at a 0.2mg dose. The increased amounts of MlP-lα and TNF-α at 0.2mg dose corresponded to the amount of β-glucan in each particle. The amounts of MCP-l or IL-12 corresponded to the concentration of sulfate (SO(4)(2-)) at a 0.2mg dose. These results suggest that inflammatory lung injury was mediated by β-glucan or SO(4)(2-), which was adsorbed into the particles, via the expression of these pro-inflammatory mediators. The results also suggest that the variations in the magnitude of inflammation of the tested Kosa samples depend on the amounts of these toxic materials.

Enhancement of Mite Allergen-Induced Eosinophil Infiltration in the Murine Airway and Local Cytokine/Chemokine Expression by Asian Sand Dust

Data on the effects of sand dust toward allergic asthma produced by indoor allergens, such as house dust mites, are not currently available. This study was undertaken to clarify the role of Asian sand dust on mite allergen, Dermatophagoides farinae (D. farinae)-induced eosinophilic inflammation in the murine lung, using sand dusts from the Maowusu Desert (Inner Mongolia) (SD-1) and the Tengger Desert (China) (SD-2). ICR mice were intratracheally administered saline; SD-1 alone; SD-2 alone; D. farinae alone; D. farinae + SD-1; and D. farinae + SD-2, 4 times at 2-wk intervals. The two sand dusts enhanced infiltration of eosinophil in the airway, along with goblet-cell proliferation related to D. farinae. The degree of eosinophil infiltration induced with SD-2 was greater than with SD-1. The SD-1, which contained higher amounts of β-glucan, increased the expression of interferon (IFN)-γ in bronchoalveolar lavage fluids (BALF) with or without D. farinae, but SD-2 did not. Synergistically or cumulatively elevated levels of interleukin (IL)-5, eotaxin, and monocyte chemotactic protein in BALF related to D. farinae were higher with D. farinae + SD-2 than with D. farinae + SD-1. These results suggest that increased cytokine and chemokines in BALF play an important role in the enhancement of eosinophil infiltration in the airway induced by D. farinae + sand dusts. The reduced eosinophil infiltration in the SD-1-treated mice could be due to suppression of Th-2 cytokine and eotaxin via interferon-γ induced by microbial materials, such as β-glucan.

Effects of phenanthraquinone on allergic airway inflammation in mice

Diesel exhaust particles (DEP) enhance allergic airway inflammation in mice (Takano et al., Am J Respir Crit Care Med 1997; 156: 36–42). DEP consist of carbonaceous nuclei and a vast number of organic chemical compounds. However, it remains to be identified which component(s) from DEP are responsible for the enhancing effects. 9,10‐Phenanthraquinone (PQ) is a quinone compound involved in DEP.

Naphthoquinone enhances antigen-related airway inflammation in mice.

The current authors have previously demonstrated that diesel exhaust particles (DEP) enhance antigen-related airway inflammation in mice. Furthermore, a recent study has shown that organic chemicals in DEP, rather than their carbonaceous nuclei, are important contributors to the aggravating effects of airway inflammation. However, the components in DEP responsible for the enhancing effects on the model remain to be identified. The current authors investigated the effects of naphthoquinone (NQ), one of the extractable chemical compounds of DEP, on antigen-related airway inflammation, local expression of cytokine proteins, and antigen-specific immunoglobulin (Ig) production in mice. Pulmonary exposure to NQ dose-dependently aggravated antigen-related airway inflammation, as characterised by infiltration of eosinophils and lymphocytes around the airways and an increase in goblet cells in the bronchial epithelium. Combined exposure to NQ and antigen enhanced the local expression of interleukin (IL)-4, IL-5, eotaxin, macrophage chemoattractant protein-1 and keratinocyte chemoattractant, compared with exposure to antigen or NQ alone. Also, NQ exhibited adjuvant activity for the antigen-specific production of IgG1 and IgG2a. These results provide the first experimental evidence that naphthoquinone can enhance antigen-related airway inflammation in vivo, and that naphthoquinone can, to some extent, partly play a role in the pathogenesis of diesel exhaust particle toxicity on the condition.

Role of Metallothionein in Antigen-Related Airway Inflammation

Metallothionein (MT) is a protein that can be induced by inflammatory mediators and participates in cytoprotection. However, its role in antigen-related inflammation remains to be established. We determined whether intrinsic MT protects against antigen-related airway inflammation induced by ovalbumin (OVA) in MT-I/II null (MT [–/–]) mice and in corresponding wild-type (WT) mice. MT (–/–) mice and WT mice were intratracheally challenged with OVA (1 µg per body) biweekly four times. Twenty-four hours after the last OVA challenge, significant increases were shown in the numbers of total cells, eosinophils, and neutrophils in bronchoalveolar lavage fluid from MT (–/–) mice than in those from WT mice. The protein level of interleukin-1β (IL-1β) was significantly greater in MT (–/–) mice than in WT mice after OVA challenge. Immunohistochemical analysis showed that the formations of 8-oxy-deoxyguanosine and nitrotyrosine in the lung were more intense in MT (–/–) mice than in WT mice after OVA challenge. These results indicate that endogenous MT is a protective molecule against antigen-related airway inflammation induced by OVA, at least partly, via the suppression of enhanced lung expression of IL-1β and via the antioxidative properties. Our findings suggest that MT may be a therapeutic target for the treatment of antigen-related airway inflammatory diseases such as bronchial asthma.