Genes and Environment最新文献

Background: Single-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, the yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized.

Results: In this report, we describe a new arabinose-inducible ss phagemid production method without helper phage infection. The newly exploited DNA derived from VCSM13 expresses the pII protein, which initiates ss DNA synthesis, under the control of the araBAD promoter. In addition, the packaging signal is deleted in the DNA to reduce the contamination of the phage-derived ss DNA. The phagemid DNA of interest, carrying the M13 origin of replication and the packaging signal, was introduced into bacterial cells maintaining the modified VCSM13 DNA as a plasmid, and the ss phagemid DNA production was induced by arabinose. The DNA recovered from the phage particles had less contamination from VCSM13 DNA, as compared to the conventional method. Moreover, we extended the method to purify the ss DNAs by using an anion-exchange column, to avoid the use of hazardous chemicals.

Conclusion: Using this combination of methods, large quantities of phagemid ss DNAs of interest can be consistently obtained.

Background: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities.

Results:  We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group.

Conclusions: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by pulmonary vascular remodeling, which can be caused by abnormal proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Several microRNAs were demonstrated to regulate the PASMC dysfunction. Our study intends to evaluate whether miR-627-5p affects cigarette smoke extract (CSE)-induced aberrant biological behaviors of PASMCs.

Methods: PASMCs was treated with CSE to create the in vitro cellular model of COPD. The viability and LDH release of PASMCs was detected by CCK-8 assay and LDH release assay. MiR-627-5p and MAP 2 K4 expression in CSE (2%)-treated PASMCs was detected by qRT-PCR. PASMC proliferation was observed under a microscope, and PASMC migration was assessed by Transwell migration assays. The binding of miR-627-5p on MAP 2 K4 was verified by dual-luciferase reporter assay. Protein levels of MAP2K4 and the PI3K/AKT signaling markers were examined by western blotting.

Results: The viability of PASMCs treated with 2% CSE reached a peak. CSE dose-dependently downregulated miR-627-5p expression in PASMCs. MiR-627-5p overexpression attenuated the CSE-induced abnormal proliferation and migration of PASMCs. However, MAP2K4 overexpression antagonized the effects of miR-627-5p on PASMC dysfunction. Importantly, miR-627-5p inhibited CSE-stimulated activation of the PI3K/AKT pathway via downregulating MAP2K4.

Conclusion: MiR-627-5p improves CSE-induced abnormal proliferation and migration of PASMCs by inhibiting MAP2K4 expression and the PI3K/AKT pathway.

Background: The repeated-dose liver micronucleus (RDLMN) assay has been well-developed and applied because of its simplicity and the ease of integration into general toxicity studies which is the preferred method from the 3R's point of view. In this assay, we observed micronucleated hepatocytes which accumulated during a rather long-term dosing period. When considering integration into general toxicity studies, the effects of age of the animals used in the micronucleus assay becomes a major issue. The effect of age on the micronucleus induction rate has been reported in bone marrow micronucleus assays, and it is considered that the decrease in cell proliferation rate due to aging is the cause of the decrease in sensitivity. A decrease in sensitivity due to aging was also reported in a liver micronucleus assay using clofibrate and the cause is considered to be a decrease in hepatocyte proliferation activity due to aging. However, no actual decrease in hepatocyte proliferation rate due to aging has been reported. In addition, there are no reports, so far, on whether similar effects of aging appear when other substances were administered. To investigate the effects of aging in the RDLMN assay, this study focused on the effects of 14-day repeated administration of DEN, a well-known genotoxic hepatocarcinogen with the hepatocyte toxicity which should cause an elevation of cell proliferation rate as a reflective regeneration.

Results: The liver micronuclei induced by DEN were equivalent between the two age groups (i.e., six and eight weeks of age at the start of dosing). In the histopathological examination for the liver, single cell necrosis, karyomegaly, and increased mitosis were observed in the hepatocytes, and the frequency and severity were increased dose-dependently. Ki-67 immunohistochemical analysis which can detect all cells in the cell cycle other than those in the G0 phase revealed dose-dependent increase of cell proliferation activity, and the difference between ages was not observed.

Conclusion: The effect of aging on the RDLMN assay could not be recognized when DEN was administered for 14 days in rats. Meanwhile, it was supported by the histopathological examination and Ki-67 immunohistochemical analysis that such an effect of aging was masked by the compensatory hepatocyte proliferation which was induced by the hepatocyte toxicity of DEN.

Bisphenol-A (BPA) is an important environmental contaminant with adverse health effects suspected to be mediated through epigenetic mechanisms. We had reported that the FLO1-dependent flocculation of transgenic yeast expressing human DNA methyltransferase (DNMT yeast) is a useful tool in epigenotoxicology studies. In this report, we have investigated the effects of BPA in the presence of metabolic activation (S-9 mix) on the transcription level of the FLO1 gene in the DNMT yeast. In the presence of metabolic activation, BPA inhibited the intensity of green fluorescence reporter protein (GFP) driven by the FLO1 promoter. A metabolite of BPA, 4-methyl-2,4-bis(p-hydroxyphenyl) pent-1-ene (MBP), also exhibited similar inhibitory effect. Furthermore, BPA in the presence of S-9 mix had only a weak while MBP had no inhibitory effects on the expression of modified GFP reporter gene under the control of FLO1 promoter with reduced CpG motifs. Aforementioned behavior was confirmed by the inhibition of flocculation as well as FLO1 gene mRNA expression. In addition, the global DNA methylation level in the human HEK293 cells was also reduced by MBP. These results indicate that BPA metabolites have inhibitory effect on DNA methylation. Our approach offers a novel in vitro method for screening for chemicals that can alter the epigenome by a mechanism dependent on their metabolic activation.