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In Vitro and In Vivo Genotoxicity Induced by Fullerene (C60) and Kaolin

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Abstract

Nanomaterials are being utilized for many kinds of industrial products, and the assessment of genotoxicity and safety of nanomaterials is therefore of concern. In the present study, we examined the genotoxic effects of fullerene (C60) and kaolin using in vitro and in vivo genotoxicity systems. Both nanomaterials significantly induced micronuclei and enhanced frequency of sister chromatid exchange (SCE) in cultured mammalian cells. When ICR mice were intratracheally instilled with these nanomaterials, DNA damage of the lungs increased significantly that of the vehicle control. Formation of DNA adducts in the lungs of mice exposed to nanomaterials were also analyzed by stable isotope dilution LC-MS/MS. 8-Oxodeoxyguanosine and other lipid peroxide related adducts were increased by 2- to 5-fold in the nanomaterial-exposed mice. Moreover, multiple (four consecutive doses of 0.2 mg per animal per week) instillations of C60 or kaolin, increased gpt mutant frequencies in the lungs of gpt delta transgenic mice. As the result of mutation spectrum analysis, G:C to C:G transversions were commonly increased in the lungs of mice exposed to both nanomaterials. In addition, G:C to A:T was increased in kaolin-exposed mice. In immunohistochemical analysis, many regions of the lungs that stained positively for nitrotyrosine (NT) were observed in mice exposed to nanomaterials. From these observations, it is suggested that oxidative stress and inflammatory responses are probably involved in the genotoxicity induced by C60 and kaolin.

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... Kaolin is a clay mineral with the chemical composition Al 2 Si 2 O 5 (OH) 4 . It is used in large quantities by numerous industries including paper production, paint, rubber, plastics, ceramics, chemicals, pharmaceuticals, and cosmetics [1]. ...
... It is used in large quantities by numerous industries including paper production, paint, rubber, plastics, ceramics, chemicals, pharmaceuticals, and cosmetics [1]. We have previously reported that kaolin particles showed genotoxic effects in in vitro and in vivo assay systems [2][3][4]. For example, kaolin nano-particles induce micronuclei in both Chinese hamster ovary (CHO) AA8 and human lung cancer A549 cell lines in micronucleus (MN) test, and DNA damages in lung of C57BL/6 J mice in comet assay [3,4]. ...
... We have previously reported that kaolin particles showed genotoxic effects in in vitro and in vivo assay systems [2][3][4]. For example, kaolin nano-particles induce micronuclei in both Chinese hamster ovary (CHO) AA8 and human lung cancer A549 cell lines in micronucleus (MN) test, and DNA damages in lung of C57BL/6 J mice in comet assay [3,4]. ...
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Introduction: Kaolin is a clay mineral with the chemical composition Al2Si2O5(OH)4. It is an important industrial material, and is also used as a white cosmetic pigment. We previously reported that fine particles of kaolin have genotoxic potency to Chinese hamster ovary CHO AA8 cells, and to the lungs of C57BL/6 J and ICR mice. In the present study, we evaluated the genotoxicity of different particle sizes of kaolin using primary normal human diploid epidermal keratinocytes and primary normal human diploid dermal fibroblasts, in addition to a CHO AA8 cell line. Findings: After 6-h treatment with kaolin micro- and nano-particles of particle sizes 4.8 μm and 0.2 μm (200 nm), respectively, the frequencies of micronucleated cells increased in a dose-dependent manner. The frequency increased 3- to 4-fold by exposure to the particles at 200 μg/mL (i.e., 31.4 μg/cm2) in all cells tested. Two-way ANOVA revealed a significant main effect of particle size, and the nano-particles tended to have a higher potency of micronucleus (MN) induction. However, the cell type did not significantly affect the MN frequencies. In addition, one-hour treatment with the kaolin particles increased DNA damage in a dose-dependent manner in a comet assay. The %tail DNA was increased 8- to 20-fold by exposure to the particles at 200 μg/mL, for all cells tested. The kaolin nano-particles had higher DNA-damaging potency than the micro-particles. Furthermore, treatment with kaolin particles dose-dependently increased the production of reactive oxygen species (ROS) in all cells. Again, we observed that kaolin nano-particles induced more ROS than the micro-particles in all cells. Conclusion: Kaolin particles demonstrated genotoxicity in primary normal human diploid epidermal keratinocytes and fibroblasts as well as in CHO AA8 cells. Although no significant difference was observed among these three types of cells, fine particles of kaolin tended to have higher genotoxic potency than coarse particles. Since studies on its genotoxicity to skin have been scarce, the findings of the present study could contribute to safety evaluations of kaolin particles when used as a white cosmetic pigment.
... Other applications of kaolin include use in the ceramics industry, cosmetics, and pharmaceuticals [1]. We have previously reported that kaolin showed genotoxic effects in in vitro and in vivo assay systems [2,3]. Recently, there have been many reports that toxicity induced by fine particles is influenced by physicochemical differences such as size [4][5][6][7][8][9]. ...
... In order to understand the reason for the differences of genotoxic potency among these kaolins, we examined incorporation rate, ROS generation and inflammatory cytokine production by in vitro systems using A549 and RAW264 cells. We have previously reported that accumulation of nitrotyrosine was observed in macrophages and alveolar epithelial cells in the lungs of mice intratracheally instilled with kaolin, thus inflammation would be partly be involved in the appearance of genotoxicity [3]. Therefore, we used these two types of cultured mammalian cells, lung epithelial cells (A549) and macrophage-like cells (RAW264). ...
... As mentioned above, ROS was considered to be an agent of nanomaterial-induced DNA damage. In fact, we previously demonstrated that kaolin induced oxidative DNA adducts such as 8-oxodG in the lungs of mice [3]. Therefore, we predicted that oxidative DNA damage in alveolar epithelial cells was induced by kaolin-phagocytized macrophage in an in vivo system. ...
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Background Kaolin 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. Results ICR mice were intratracheally instilled with the kaolins (0.05 and 0.2 mg/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. Conclusion From 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.
... The gpt mutagenesis assay was performed according to the previously described method. [3][4][5][6][7] The mutation spectra of the gpt coding sequence were assessed by PCR and direct sequencing. Briefly, a 739-bp DNA fragment containing gpt was amplified by PCR as described previously, [3][4][5][6]15 and the sequencing analysis was done at Takara Bio (Mie, Japan). ...
... [3][4][5][6][7] The mutation spectra of the gpt coding sequence were assessed by PCR and direct sequencing. Briefly, a 739-bp DNA fragment containing gpt was amplified by PCR as described previously, [3][4][5][6]15 and the sequencing analysis was done at Takara Bio (Mie, Japan). ...
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Engineered nanomaterials (ENMs) are now used in a wide variety of fields, and thus their safety should urgently be assessed and secured. It has been suggested that inflammatory responses via the phagocytosis of ENMs by macrophages is a key mechanism for their genotoxicity. The present study was conducted to establish a mechanism-based assay to evaluate the genotoxicity of ENMs under conditions simulating an in vivo situation, featuring a co-culture system of murine lung resident cells (GDL1) and immune cells (RAW264.7). GDL1 were cultured with or without RAW264.7, exposed to a multi-walled carbon nanotube (MWCNT), and then analyzed for mutagenicity and underlying mechanisms. Mutation frequencies induced in GDL1 by the MWCNT were significantly greater with the co-existence of RAW264.7 than in its absence. Mutation spectra observed in GDL1 co-cultured with RAW264.7 were different from those seen in GDL1 cultured alone, but similar to those observed in the lungs of mice exposed to the MWCNT in vivo. Inflammatory cytokines, such as IL-1β and TNF-α, were produced from RAW264.7 cells treated with the MWCNT. The generation of reactive oxygen species and the formation of 8-oxodeoxyguanosine in GDL1 exposed to the MWCNT were greater in the co-culture conditions than in the single culture conditions. Based on these findings, it is indicated that inflammatory responses are involved in the genotoxicity of MWCNT, and that the presently established, novel in vitro assay featuring a co-culture system of tissue resident cells with immune cells is suitable to evaluate the genotoxicity of ENMs.
... The prominent mutation types induced by MGT were a G:C to A:T transition followed by a G:C to T:A transversion. In contrast, G:C to C:G transversion was not more frequent with MGT treatment, whereas this type of transversion is commonly increased by fullerene, kaolin and multi-walled carbon nanotubes (MWCNTs) [21][22][23]. From this observation, it is suggested that mechanisms leading to the induction of mutations in mice lungs by MGT might be somehow different from other particles, such as fullerene, kaolin and MWCNTs. In general, the G:C to A:T transition and G:C to T:A transversion have commonly been observed in spontaneous mutants. ...
... Lambda EG10 phages were rescued using Transpack Packaging Extract (Stratagene). The gpt mutagenesis assay was performed according to previously described methods [21][22][23]36]. ...
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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.
... A reliable marker of oxidative damage to biomacromolecules by ROS is the level of long-lived active forms of proteins and 8-oxoguanine in DNA [71][72][73][74][75][76]. Our study showed that borosiloxane does not affect the level of long-lived active forms of proteins and 8-oxoguanine in DNA in vitro. ...
Article
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A technology for producing a nanocomposite based on the borsiloxane polymer and chemically unmodified fullerenes has been developed. Nanocomposites containing 0.001, 0.01, and 0.1 wt% fullerene molecules have been created. It has been shown that the nanocomposite with any content of fullerene molecules did not lose the main rheological properties of borsiloxane and is capable of structural self-healing. The resulting nanomaterial is capable of generating reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals in light. The rate of ROS generation increases with an increase in the concentration of fullerene molecules. In the absence of light, the nanocomposite exhibits antioxidant properties. The severity of antioxidant properties is also associated with the concentration of fullerene molecules in the polymer. It has been shown that the nanocomposite upon exposure to visible light leads to the formation of long-lived reactive protein species, and is also the reason for the appearance of such a key biomarker of oxidative stress as 8-oxoguanine in DNA. The intensity of the process increases with an increase in the concentration of fullerene molecules. In the dark, the polymer exhibits weak protective properties. It was found that under the action of light, the nanocomposite exhibits significant bacteriostatic properties, and the severity of these properties depends on the concentration of fullerene molecules. Moreover, it was found that bacterial cells adhere to the surfaces of the nanocomposite, and the nanocomposite can detach bacterial cells not only from the surfaces, but also from wetted substrates. The ability to capture bacterial cells is primarily associated with the properties of the polymer; they are weakly affected by both visible light and fullerene molecules. The nanocomposite is non-toxic to eukaryotic cells, the surface of the nanocomposite is suitable for eukaryotic cells for colonization. Due to the combination of self-healing properties, low cytotoxicity, and the presence of bacteriostatic properties, the nanocomposite can be used as a reusable dry disinfectant, as well as a material used in prosthetics.
... 170 Genotoxicity has also been demonstrated by some authors, although the mechanism is not clear (probably due to oxidative stress). 171 However, in another study, genotoxicity was assessed in a bacterial reverse mutation assay (Ames test) and a chromosomal aberration test, with no significant increase being found in the frequency of chromosomal aberration. 164 The use of fullerenes in cosmetics has implications for the human and aquatic environment, as fullerene molecules in creams could be released into wastewater. ...
Chapter
New nanomaterials, both biodegradable and non-biodegradable, are finding many applications in various fields including, among others, energy, food, cosmetics, medicine, textiles, construction and computing. However, because of their small size, nanostructures (Ns) can enter live organisms through unsuspected routes and interact with their biological systems. The intended or unintended entry of nanostructures can induce toxicity at several levels, which should be carefully studied. Moreover, it is important to monitor work places where staff can experience (acute or chronic) exposure to large amounts of Ns, as well as to analyse their potential ecotoxicity. This chapter provides an overview of the potential toxicity of Ns, distinguishing between biodegradable and non-biodegradable nanostructures.
... To investigate nitric oxide production after nanoparticle exposure, immunohistochemical staining of inflammation factors, such as inducible NO synthase (iNOS) and nitrotyrosine (NT), in the lungs of gpt delta mice treated with MWCNTs were examined, using a procedures reported previously (Totsuka et al. 2010;Porter et al. 2002). ...
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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.
... 59 DNA damage and altering nucleotide base play an important role in DNA mutations, 60,61 as there is a possibility that fullerene interactions could oxidatively damage DNA and cause a disease in the long run. 2 For example, the application of fullerene significantly induced micronuclei and enhanced frequency of sister chromatid exchange and increased the frequency of transversion in cultured mammalian cells. 62 Janne et al. reported there was an increased mRNA expression of 8-oxoguanine DNA glycosylase in the liver of C 60 fullerene-treated rats, but no significant increase in repair activity was found. 63 Fullerenol binding has the prospective effect on the inhibition of enzymes involved in DNA replication, transcription, and repair, resulting in misfunction of DNA and consequently increasing the mutation rates. ...
Article
Buckminsterfullerene (C(60)) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C(60) could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNA-C(60) binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C(60) aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 70-85 °C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C(60) on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.
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This study aimed at comparative examining of the interactions between conventionally used clay and carbon nanomaterials and human lung adenocarcinoma cells (A549 cells). The following platy and tubular nanomaterials were tested: carbon nanoparticles, i.e. multi-walled carbon nanotubes (MWCNTs) and graphene oxide nanosheets (GO) as well as nanoclays, i.e. halloysite nanotubes (HNTs) and kaolinite nanosheets (Kaol). Nanoparticle physicochemical properties and their internalization into cells were examined using dynamic light scattering as well as atomic force, 3D laser scanning confocal and darkfield hyperspectral microscopies. Biological aspects of the nanomaterial-cell interaction included assessment of cellular toxicity, DNA damage, metabolic activity, and physical parameters of the cells. Regardless of a shape, carbon nanomaterials demonstrated cell surface adsorption, but negligible penetration into cells compared to nanoclays. However, carbon nanomaterials were found to be the most toxic for cells as probed by the MTS assay. They also turned out to be the most genotoxic for cells compared to nanoclays as revealed by the DNA-Comet assay. GO significantly increased the fraction of apoptotic cells and was the most cytotoxic and genotoxic nanomaterial. Comparison of flow cytometry and MTS data indicated that a cytotoxic effect of MWCNTs was not associated with increased cell death, but was rather due to a decrease in cell metabolic activity and/or proliferation. Finally, no significant effect of the shape of the tested nanomaterials on their internalization and cytotoxicity was revealed.
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Recently, 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. In 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. Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.
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In vitro work in this laboratory has identified new DNA lesions resulting from further oxidation of a common biomarker of oxidative damage, 8-oxo-7,8-dihydroguanine (OG). The major product of oxidation of OG in a nucleoside, nucleotide, or single-stranded oligodeoxynucleotide using metal ions that act as one-electron oxidants is the new nucleoside derivative spiroiminodihydantoin (Sp). In duplex DNA an equilibrating mixture of two isomeric products, guanidinohydantoin (Gh) and iminoallantoin (Ia), is produced. These products are also formed by the overall four-electron oxidation of guanosine by photochemical processes involving O(2). DNA template strands containing either Sp or Gh/Ia generally acted as a block to DNA synthesis with the Klenow exo(-) fragment of pol I. However, when nucleotide insertion did occur opposite the lesions, only 2'-deoxyadenosine 5-triphosphate and 2'-deoxyguanine 5-triphosphate were used for primer extension. The Escherichia coli DNA repair enzyme Fpg was able to remove the Sp and Gh/Ia lesions from duplex DNA substrates, although the efficiency was depended on the base opposite the lesion.
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The low redox potential of 8-oxo-7,8-dihydroguanine (OG), a molecule regarded as a marker of oxidative damage in cells, makes it an easy target for further oxidation. Using a temperature-dependent method of synthesis, the oxidation products of OG, guanidinohydantoin (Gh) and/or its isomer iminoallantoin (Ia) as well as spiroiminodihydantoin (Sp), have been site-specifically incorporated into DNA oligomers. Single nucleotide insertion and primer extension experiments using Escherichia coli Kf exo(-) DNA polymerase were carried out under "standing start" and "running start" conditions in various sequence contexts. dAMP and dGMP were found to be inserted opposite these OG oxidation products. Steady-state kinetic studies show that the Gh/Ia.G base pair yields a lower K(m) value compared to the Sp.G pair or X.A (X = Gh/Ia or Sp). Running start experiments using oxidized and unoxidized OG-containing templates showed enhanced full extension in the presence of all four dNTPs. A sequence preference for efficiency of extension was found when Gh/Ia and Sp are present in the DNA template, possibly leading to primer misalignment. Full extension is more efficient for the templates containing two Gs immediately 3' to the lesions compared to two As. Although these lesions cause a significant block for DNA elongation, results show that they are more easily bypassed by the polymerase when situated in the appropriate sequence context. UV melting studies carried out on duplexes mimicking the template/primer systems were used to characterize thermal stability of the duplexes. These experiments suggest that both Gh/Ia and Sp destabilize the duplex to a much greater extent than OG, with Sp being most severe.
Chapter
The mechanism by which asbestos and other mineral fibers cause cancer remains unknown despite intensive investigation. The most carcinogenic forms of asbestos contain iron and catalyze many of the same reactions that iron does. In fact, there is increasing evidence to suggest that iron is responsible for the biochemical reactivity of asbestos in vitro. Iron appears to be responsible for asbestos-dependent 02 consumption [Aust and Lund (1991); Lund and Aust (1991)], OH formation [Weitzman and Graceffa (1984); Gulumian and Van Wyk (1987); Zalma et al. (1987); Kennedy et al. (1989); Aust and Lund (1991)], lipid peroxidation [Weitzman and Weitberg (1985); Turver and Brown (1987); Goodlick et al. (1989)], induction of deoxyribonuclease-S1 sensitive sites [Turver and Brwon (1987)], and induction of DNA single-strand breaks [Lund and Aust (1992)]. Iron may also play a role in one mechanism of phagocytosis of asbestos fibers [Hobson et al. (1990)]. Desferrioxamine B, an iron-specific chelator, reduced the cytotoxicity of asbestos to a variety of different cultured cell types, suggesting that iron may be involved in the cytotoxicity of asbestos [Goodlick and Kane (1986); Shatos et al. (1987); Garcia et al. (1988); Kamp et al. (1990); Goodlick and Kane (1990)].
The electrochemical oxidation of guanosine and 8-hydroxyguanosine has been studied in phosphate buffers at glassy carbon and pyrolytic graphite electrodes. On the basis of the electrochemical, spectroelectrochemical and product analysis, it has been concluded that oxidation of guanosine proceeds via the formation of 8-hydroxyguanosine. The initial electrode reaction has been deduced to involve a Ie, 1H+ step to give a free radical which on further oxidation gives 8-hydroxyguanosine. The 2e, 2H+ oxidation of 8-hydroxyguanosine rapidly gives a diimine species which undergoes a series of chemical reactions to give urea riboside, 2-amino-4,5,6-trioxypyrimidine and a dimer at pH 3.0 and 5-guanidinohydantoin at pH 7.0. The peroxidase catalysed oxidation of 8-hydroxyguanosine was also found to proceed by a mechanism identical to electrochemical oxidation. Intracranial injection of a single dose of the oxidation product of guanosine (dimer) in albino mice caused nephritis with edema and hence was toxic in nature.
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DNA adducts are produced both exogenously and endogenously via exposure to various DNA-damaging agents. Two lipid peroxidation (LPO) products, 4-oxo-2(E)-nonenal (4-ONE) and 4-oxo-2(E)-hexenal (4-OHE), induce substituted etheno-DNA adducts in cells and chemically treated animals, but the adduct levels in humans have never been reported. It is important to investigate the occurrence of 4-ONE- and 4-OHE-derived DNA adducts in humans to further understand their potential impact on human health. In this study, we conducted DNA adductome analysis of several human specimens of pulmonary DNA as well as various LPO-induced DNA adducts in 68 human autopsy tissues, including colon, heart, kidney, liver, lung, pancreas, small intestine, and spleen, by liquid chromatography tandem mass spectrometry. In the adductome analysis, DNA adducts derived from 4-ONE and 4-OHE, namely, heptanone-etheno-2'-deoxycytidine (HεdC), heptanone-etheno-2'-deoxyadenosine (HεdA), and butanone-etheno-2'-deoxycytidine (BεdC), were identified as major adducts in one human pulmonary DNA. Quantitative analysis revealed 4-ONE-derived HεdC, HεdA, and heptanone-etheno-2'-deoxyguanosine (HεdG) to be ubiquitous in various human tissues at median values of 10, 15, and 8.6 adducts per 10(8) bases, respectively. More importantly, an extremely high level (more than 100 per 10(8) bases) of these DNA adducts was observed in several cases. The level of 4-OHE-derived BεdC was highly correlated with that of HεdC (R(2) = 0.94), although BεdC was present at about a 7-fold lower concentration than HεdC. These results suggest that 4-ONE- and 4-OHE-derived DNA adducts are likely to be significant DNA adducts in human tissues, with potential for deleterious effects on human health.
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Sunlight is regarded to be a cause of skin cancer, though the mechanisms underlying the causation are still unclear. The genotoxic effects of sunlight are believed to be induced by pyrimidine photoproducts produced by the action of the UV portion of sunlight. However, it is not clear whether these pyrimidine modifications are the sole sources for the mutations. In the present study, we have analyzed the mutagenic potential of sunlight on the lacZ alpha region of single-stranded DNA phage M13mp2 using an SOS-deficient recA- strain and an SOS-induced rec+ strain of Escherichia coli as hosts. Exposure to sunlight caused mutations; approximately 10-fold increases in the mutation frequency were observed with the use of both hosts. When SOS functions were induced in the host CSH50, the mutation frequencies increased another 10-fold over those obtained with the host lacking the SOS functions. DNA sequences of the mutants were analyzed by automated DNA sequencers. Sequence changes were identified in 53 mutants from the mutant DNAs obtained using NR9099 as host and in 78 mutant samples obtained using UV-treated CSH50. Most of the mutations were transversions of guanine, either G to C or G to T. Furthermore, 59% of the identified sequence changes in the SOS- host and 40% of those in the SOS-induced host were G-to-C transversions. These transversions may be caused by unidentified guanine damages or by the effects of damage at pyrimidines distal from guanines to be mutated.
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A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe3+/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C-->C:G (28 cases) and G:C-->T:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5'-PuGA-3' was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5'PyGN3'. G:C-->T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:C-->C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.
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Oxidative damage of DNA by endogenously generated oxygen radicals contributes to the mutagenic process. Hydroxy, alkoxy, and peroxy radicals all have the potential to react with DNA, giving rise to strand breaks and potentially mutagenic oxidative base damage. Although reactions of the hydroxy radical with DNA have been well studied, far less is known about the reactivities of these other radicals with DNA and their mutation-inducing potential. Frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation were measured by glyoxal gel electrophoretic analysis. We report the spectrum of mutations induced in Escherichia coli upon transfection with peroxy radical treated DNA carrying the lacZ alpha gene as a reporter. Transfection of DNA exposed to micromolar amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS-inducible cells. Sequencing analysis of DNA isolated from mutants showed that among base substitution mutants 88% consisted of transversions at G, with a nearly equal number of G --> C and G --> T mutants. Transition mutations were rarely detected, in contrast to control experiments. Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed to any detectable degree. The oxidative G lesions are sensitive to digestion by the Fpg protein. We were unable to detect the formation of 8-oxo-dG by HPLC/electrochemical analysis of peroxy radical oxidation of dG, suggesting that the G --> T transversions were not caused by this base lesion.
Article
We report the identification of a mouse kidney epithelial cell line (K435) in which G:C-->C:G transversion mutations occur at an elevated rate and are the predominant spontaneous events observed at the selectable Aprt locus. Of three genotoxins tested, ultraviolet radiation (UV), ionizing radiation, and hydrogen peroxide, only UV exposure was able to alter the spectrum of small mutational events. To determine if the G:C-->C:G mutator phenotype was due to a deficiency in the mismatch repair pathway, the K435 cells were tested for resistance to 6-thioguanine, cisplatin, and MNNG. Although the K435 cells were as resistant to 6-thioguanine and cisplatin as Pms2 and Mlh1 null kidney cells, they were hypersensitive to MNNG. Moreover, the K435 cells do not exhibit microsatellite instability, a hallmark of mismatch repair deficiency. These results suggest that a novel mechanism, which does not include a classical deficiency in mismatch repair, accounts for the G:C-->C:G mutator phenotype.
Article
In previous reports from this study, measurements of pulmonary inflammation, bronchoalveolar lavage cell cytokine production and nuclear factor-kappa B activation, cytotoxic damage, and fibrosis were detailed. In this study, we investigated the temporal relationship between silica inhalation, nitric oxide (NO), and reactive oxygen species (ROS) production, and damage mediated by these radicals in the rat. Rats were exposed to a silica aerosol (15 mg/m(3) silica, 6 h/day, 5 days/wk) for 116 days. We report time-dependent changes in 1) activation of alveolar macrophages and concomitant production of NO and ROS, 2) immunohistochemical localization of inducible NO synthase and the NO-induced damage product nitrotyrosine, 3) bronchoalveolar lavage fluid NO(x) and superoxide dismutase concentrations, and 4) lung lipid peroxidation levels. The major observations made in this study are as follows: 1) NO and ROS production and resultant damage increased during silica exposure, and 2) the sites of inducible NO synthase activation and NO-mediated damage are associated anatomically with pathological lesions in the lungs.
Article
Guanosine labeled with 15N at N1, amino, and N7 and 13C at either C2 or C8 was oxidized by Rose Bengal photosensitization (singlet oxygen) in buffered aqueous solution. At pH > 7, spiroiminodihydantoin was the major product, while at pH < 7, guanidinohydantoin (Gh) was the principal product. 15N and 13C NMR studies confirmed that Gh was formed as a mixture of slowly equilibrating diastereomers. Experiments conducted in H218O indicated that Gh and Sp each contained one oxygen atom derived from O2 and one from H2O. Tandem mass spectrometry was used to identify the C4 carbonyl of Gh as the one labeled with 18O, supporting a mechanism involving attack of water at C5 of a dehydro-8-oxoguanosine intermediate.
Article
Cellular DNA is exposed to a variety of exogenous and endogenous mutagens. A complete understanding of the importance of different types of DNA damage requires knowledge of the specific molecular alterations induced by different types of agents in specific target tissues in vivo. The gpt delta transgenic mouse model provides the opportunity to characterize tissue-specific DNA alterations because small and large deletions as well as base substitutions can be analyzed. Here, we summarize the characteristics of intrachromosomal deletions and base substitutions induced by ionizing radiation in liver and spleen, ultraviolet B (UVB) radiation in epidermis, mitomycin C (MMC) in bone marrow, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in colon, and aminophenylnorharman (APNH) in liver of gpt delta mice. Carbon-ion radiation, UVB, and MMC induced large deletions of more than 1 kb. About half of the large deletions occurred between short direct-repeat sequences and the remainder had flush ends, suggesting the involvement of nonhomologous end joining of double-stranded breaks (DSBs) in DNA. UV photoproducts and interstrand crosslinks by MMC may block DNA replication, thereby inducing DSBs. In contrast, PhIP and APNH mainly generated 1 bp deletions in runs of guanine bases. As for base substitutions, UVB and MMC induced G:C-->A:T transitions at dipyrimidine sites and tandem base substitutions at GG sites, respectively. PhIP and APNH induced G:C-->T:A transversions. Translesion DNA synthesis across the lesions, i.e., UV photoproducts, intrastrand crosslinks by MMC, and guanine adducts by the heterocyclic amines, may be involved in the induction of base substitutions. These results indicate the importance of sequence information to elucidate the mechanisms underlying deletions and base substitutions induced in vivo by environmental mutagens.
Article
Many oxidizing agents induce G-C to T-A and G-C to C-G transversions, and the frequency largely depends on the oxidative conditions. Guanine is the most oxidizable base among natural bases. The typical oxidative lesion product 8-oxoguanine (8-oxoG) is responsible for G-C to T-A transversion but not for G-C to C-G transversion, and 8-oxoG is more readily oxidized than guanine because of its lowered ionization potential. Recently, imidazolone (Iz), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) have been demonstrated as oxidative lesion products of guanine and 8-oxoG, which could be responsible for G-C to C-G transversions by forming specific base pair formations.
Article
Guanine is the most oxidizable base among natural bases. 8-Oxoguanine (8-oxoG) is the typical oxidative product, but the amount of 8-oxoG does not directly reflect the strength of oxidative stress. Imidazolone, oxazolone and guanidinohydantoin are oxidative products of guanine and 8-oxoG. Here, we investigated enzymatic reactions with human DNA polymerase eta on these lesions.
Article
Viability, cell cycle effects, genotoxicity, reactive oxygen species production, and mutagenicity of C(60) fullerenes (C(60)) and single-walled carbon nanotubes (SWCNT) were assessed in the FE1-Mutatrade markMouse lung epithelial cell line. None of these particles induced cell death within 24 hr at doses between 0 and 200 microg/ml or during long-term subculture exposure (576 hr) at 100 microg/ml, as determined by two different assays. However, cell proliferation was slower with SWCNT exposure and a larger fraction of the cells were in the G1 phase. Exposure to carbon black resulted in the greatest reactive oxygen species generation followed by SWCNT and C(60) in both cellular and cell-free particle suspensions. C(60) and SWCNT did not increase the level of strand breaks, but significantly increased the level of FPG sensitive sites/oxidized purines (22 and 56%, respectively) determined by the comet assay. The mutant frequency in the cII gene was unaffected by 576 hr of exposure to either 100 microg/ml C(60) or SWCNT when compared with control incubations, whereas we have previously reported that carbon black and diesel exhaust particles induce mutations using an identical exposure scenario. These results indicate that SWCNT and C(60) are less genotoxic in vitro than carbon black and diesel exhaust particles.