It is not widely known how the developing brain responds to extrinsic damage, although the developing brain is considered to be sensitive to diverse environmental factors including DNA-damaging agents. This paper reviews the mechanisms of neurotoxicity induced in the developing brain of mice and rats by six chemicals (ethylnitrosourea, hydroxyurea, 5-azacytidine, cytosine arabinoside, 6-mercaptopurine and etoposide), which cause DNA damage in different ways, especially from the viewpoints of apoptosis and cell cycle arrest in neural progenitor cells. In addition, this paper also reviews the repair process following damage in the developing brain.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs) and similar compounds are toxic to animals and humans. Based on a yeast reporter system, AhR-activating ligands similar in concentration to 2 ng/l of TCDD were detected in two canal waters in Guangzhou, China. In this study, a three-generation experiment was conducted to assess the reproductive and developmental risks associated with these waters in C57BL/6J female mice, including female reproduction, pup indices, reproductive hormone levels, and levels of AhR, ARNT, and CYP1A2 in the uterus. Similar reproductive toxic effects were produced in the offspring of mice that drank the canal water as would occur if they drank 2 ng/l/day TCDD. The major reproductive indices that were affected included mating time and gestation length over all the generations. A striking finding is the TCDD (2 ng/l) and the water samples significantly reduced Day 4 pup survival rates in the F2 and F3. Both TCDD exposure and drinking canal water decreased estradiol-17β (E2) levels in the multiparous females and decreased follicle-stimulating hormone (FSH), luteinizing hormone (LH) and E2 levels in the virgin females. Immunochemical staining revealed that the AhR and CYP1A2 positive signals were enhanced, and the ARNT positive signal was weakened in the uteri of mice drinking water with TCDD (2 ng/l) and the canal water samples. These results imply that the canal water contains AhR ligands that could induce similar toxic effects as do low levels of TCDD. Exposure to these contaminants can significantly impair the reproductive health of female mice. Considering this canals are open directly to Pearl River, whether these effects could be caused in human reproduction and development warrants further study.
Selenium (Se) is an essential micronutrient because it forms the active center of selenoenzymes/selenoproteins in the form of selenocysteine. Another biological significance of Se is that it detoxifies inorganic mercury (iHg) by directly interacting with it. Recently, a novel selenometabolite, selenoneine (2-selenyl-N,N,N-trimethyl-L-histidine), was identified in several marine animals. However, its biological significance is still unclear. In this study, the ability of selenoneine to form a complex with iHg and methyl Hg (MeHg) was evaluated in vitro. Whereas selenite serving as the positive control reacted with iHg by direct interaction after being converted into selenide by endogenous reductants, such as glutathione (GSH), selenoneine did not interact with iHg or MeHg in the liver homogenate of marine turtle. This indicates that selenoneine may not play a role in the detoxification of Hg.
Excessive production of transforming growth factor-β1 (TGFβ1) plays an important role in lung fibrosis, in which the differentiation of fibroblasts into myofibroblasts is a key process. Increased formation of reactive oxygen species (ROS) induced by TGFβ1 is a common pathological feature of fibrosis. In the present study, probucol and lovastatin, which are therapeutics used for hyperlipidemia and proposed to act as anti-oxidants, were examined in terms of their effect on TGFβ1-induced formation of ROS and expression of α-smooth muscle actin (αSMA), a myofibroblast marker, in human fetal lung fibroblasts (HFL1 cells). The effects of anti-oxidative enzymes and reagents including N-acetyl-L-cysteine, α-tocopherol, and lecithinized-superoxide dismutase (SOD) on TGFβ1-induced expression of αSMA were also examined. Treatment with probucol (30 µM) and lovastatin (1 µM and 3 µM), in addition to lecithinized-SOD, significantly inhibited the TGFβ1-induced formation of ROS and αSMA. Other anti-oxidants including N-acetyl-L-cysteine had marginal effects on αSMA expression under the conditions. Probucol and lovastatin, established therapeutics, may be worth trying in patients with both lung fibrosis and hypercholesterolemia.
It has recently been reported that activation of P2Y1 receptor, one of the purine receptors, by extracellular nucleotides induces cytoprotection against oxidative stress. In this study, we examined the protective effect of ATP on the cell damage in human epidermal keratinocyte HaCaT cells exposed to H2O2 via the P2Y receptor-mediated induction of intracellular antioxidants. The cells were damaged by exposure to H2O2 in a dose- and time-dependent manner. The damage induced by 7.5 mM H2O2 was blocked by pretreatment of the cells with ATP (1-10 µM). The protective effect of ATP was significantly reduced by P2Y receptor antagonists. Exogenously added ATP induced various intracellular antioxidants, including thiol-containing proteins, Cu/Zn superoxide dismutase (SOD) and thioredoxin-1, in HaCaT cells. In conclusion, it was found that ATP protected the cells from the H2O2-induced cell damages via the P2Y receptor-mediated induction of intracellular antioxidants.
Atelocollagen (AC), a biomaterial with low antigenicity and high bioaffinity, has been widely used in implantable materials in clinical practice. Preclinical studies have demonstrated that AC is a potential drug carrier for local and systemic delivery of cytokines, growth factors, plasmid DNA, small interfering RNA, and microRNA. AC is also believed to have low systemic toxicity on the basis of the safety of implant usage; however, this is not enough determined. Therefore, we performed whole genome expression profiling in mouse liver after systemic administration of AC or the cationic liposome carrier DOTAP/cholesterol (LP) and compared the changes of gene expressions associated with hepatotoxicity. Microarray analysis revealed that systemic LP administration significantly increased expression of toxicity-related genes, i.e., those for lipocalin-2, cyclin-dependent kinase inhibitor 1A, serum amyloid A isoforms, chemokine ligands, and granzyme B. Alternatively, AC administration did not alter the expression of any of these genes. Further gene ontology (GO) enrichment analysis highlighted the characteristic annotations extracted from genes upregulated after LP administration, and most of them were related to toxicity annotations such as immune response, inflammatory response, and apoptosis induction. In contrast, GO enrichment analysis of genes induced after AC administration revealed that only three annotations, all of which were unrelated to toxicity. These findings indicate that AC is potentially far less hepatotoxic than LP after systemic administration, suggesting that AC may be an excellent biomaterial for nontoxic drug delivery system carriers.
Subchronic toxicity of a horseradish extract (HRE), consisting mainly of a mixture of allyl isothiocyanate (AITC) and other isothiocyanates, was investigated with administration at concentrations of 0, 0.0125, 0.025 and 0.05% of HRE in drinking water for 13 weeks to male and female F344 rats. For comparison, treatment with 0.0425% of AITC was similarly performed. Body weight gain was reduced in the 0.05% HRE and AITC males as compared to the 0% controls, and the cause was considered at least partly related to decreased water consumption due to the acrid smell of the test substance and decreased food consumption. Serum biochemistry demonstrated increased urea nitrogen in 0.025 and 0.05% HRE and AITC males and 0.0125-0.05% HRE and AITC females, along with decreased total cholesterol in 0.0125-0.05% HRE females. On histopathological assessment, papillary/nodular hyperplasia of bladder mucosa was observed in 0.05% HRE and AITC males and females, in addition to simple mucosal hyperplasia found in all treated groups. Based on the above findings, no-observed-adverse-effect levels (NOAELs) were estimated to be below 0.0125% of HRE for both males and females, corresponding to 9.4 and 8.0 mg/kg body weight/day, respectively, and there appeared to be comparable toxicological properties of HRE to AITC, such as the inductive effect of significant proliferative lesions in the urinary bladder.
Indole-3-carbinol (I3C) has a liver tumor promoting activity in rats, and is also known as a cytochrome p450 1A (CYP1A) inducer. The generation of reactive oxygen species (ROS) resulting from CYP1A induction due to I3C, is probably involved in the tumor promotion. To clarify whether ROS generation contributes to I3C’s induction of hepatocellular altered foci, partially hepatectomized rats were fed a diet containing 0.5% of I3C for 8 weeks with or without 0.3% N-acetyl-L-cysteine (NAC), an antioxidant, in their drinking water after N-diethylnitrosamine (DEN) initiation. Immunohistochemical analysis showed that the glutathione-S-transferase placental form (GST-P) positive foci promoted by I3C were suppressed by the administration of NAC. The mRNAs of members of the phase II nuclear factor, erythroid derived 2, like 2 (Nrf2) gene batteries, whose promoter region is called as antioxidant response element (ARE), were down-regulated in the DEN-I3C-NAC group compared to the DEN-I3C group, but Cyp1a1 was not suppressed in the DEN-I3C-NAC group compared to the DEN-I3C group. There was no marked difference in production of microsomal ROS and genomic 8-hydroxy-2′-deoxygunosine (8-OHdG) as an oxidative DNA marker between the DEN-I3C-NAC and DEN-I3C groups, while mapkapk3 and Myc were decreased by the NAC treatment. These results indicate that oxidative stress plays an important role for I3C’s tumor promotion, and NAC suppresses induction of hepatocellular altered foci with suppressed cytoplasmic oxidative stress.
Gatifloxacin has been associated with increased risks of hypoglycemic and hyperglycemic side effects. In order to understand the molecular mechanism of gatifloxacin induced deregulation of glucose metabolism, a combination of comparative and chemical proteomic approaches were employed using yeast as a model system. Differential protein expression studies using two dimensional electrophoresis and mass spectrometry reveal that gatifloxacin deregulates the expression of key enzymes involved in glucose metabolism. Furthermore, affinity chromatography and LC-MSE analysis led to identification of enolase, as one of the key gatifloxacin binding proteins. Fluorescence spectrometric studies confirmed that the gatifloxacin indeed binds to enolase. Role of enolase in regulation of gatifloxacin induced dysglycemic effect is discussed.
Recently, we identified that olive leaf extract (OLE) prevents lead (Pb)-induced abnormalities in behavior and neurotransmitters production in chronic Pb exposure in rats. The aim of the present study was to provide additional evidence that OLE acts as an anti-apoptotic, anti-inflammatory, and antioxidant mediator in Pb exposed rats. 4-weeks old Wistar rats were exposed or not to 250 mg/l Pb for 13-weeks and then exposed to tap water containing or not 0.1% OLE for additional 2-weeks. Atomic absorption spectrophotometry showed significantly elevated Pb levels in the hippocampus and serum and reaches 5 and 42 μg/mg tissue, respectively. In the hippocampus, the examination of markers of apoptosis and inflammation revealed an increase in caspase-3 activity and DNA fragmentation as well as tumor necrosis factor alpha, interleukin-1 beta and prostaglandin E2 in Pb-exposed rats. In addition, our findings showed that Pb induced 4-hydroxynonenal production and inhibited antioxidant-related enzyme activity, such as glutathione-S-transferase as wells as energy metabolism-related enzyme activity, such as NADP-isocitrate dehydrogenase and glucose transporter. Upon examination of signaling pathways involved in apoptosis process, we found that Pb induced p38 mitogen activated protein kinase (MAPK) and Akt phosphorylation, but in contrast, inhibited that of ERK1/2. Interestingly, OLE administration diminished tissue Pb deposition and prevented all Pb effects. In the frontal cortex, our data also showed that OLE-abolished Pb-induced caspase-3 activity and DNA fragmentation. Collectively, these data support the use of OLE by traditional medicine to counter Pb neurotoxicity.
Polyethylene glycol 400 (PEG 400) is widely used with a variety of pharmaceutical formulations, and is often added to dosing formulations in preclinical toxicity studies. The aim of the present study was to characterize the effects of PEG 400 on the rat gastrointestinal tract. Three dosage levels (5, 50 or 100 v/v%) of PEG 400 were administered at a volume of 5 ml/kg/day by gavage for 15 days to the rats (5 males and 5 females in each group). At the end of the treatment, the whole lengths of gastrointestinal tracts were examined pathologically. Although there were no gross abnormalities at necropsy, the histopathological examination revealed several changes localized to the stomach mucosa, but not in the intestine. The changes consisted of infiltration of eosinophils and globule leukocytes, increased in the height of the entire mucosal layer, elongation of the surface mucous epithelial and mucous neck cell layers with increased intracellular mucous in the glandular stomach, and the spongiosis (intercellular edema) of the squamous epithelium in the forestomach. These changes near the limiting ridge tended to increase in severity and extent in a dose-dependent manner. These results suggest that repeated oral administration of concentrated PEG 400 can easily induce the mucosal changes in the stomach of the rats.
1,2-Naphthoquinone (1,2-NQ) is an electrophile found in the atmosphere, which reacts readily with protein nucleophiles to form a stable protein adduct. Peroxiredoxin 6 (Prdx6) is predominantly expressed in lung tissue and functions in antioxidant defense by facilitating the repair of damaged cell membranes via reduction of peroxidized phospholipids. In the present study, human A549 pulmonary epithelial cells were exposed to 1,2-NQ to explore whether 1,2-NQ can bind covalently to Prdx6, thereby disrupting its catalytic activity. Two-dimensional SDS/PAGE followed by western blot analysis with a specific antibody against 1,2-NQ showed that Prdx6 was covalently modified by 1,2-NQ. Using purified human Prdx6, it was found that 1,2-NQ bound covalently to Prdx6 through Cys47, Lys144 and Cys91, resulting in a significant reduction in phospholipase A2 activity. These results suggest that arylation of Prdx6 by 1,2-NQ may, at least in part, be involved in the cellular toxicity induced by 1,2-NQ.
It has been shown that pulmonary exposure to diesel exhaust particles (DEP) disrupts the immune system, presenting as exacerbating effects on allergic manifestations (e.g., allergic asthma). However, since a model inhalation system has not been developed, the impact of nano-level DEP on health has not been satisfactorily investigated. Our institute (the National Institute for Environmental Studies) established an “environmental nanoparticle exposure system applied in animals” in 2005 and since then, we have explored the health effects of exposure to these types of agent. The present study was conducted to investigate the in vitro effects of nanoparticle-rich DEP (NRDEP) on primary splenocytes from atopy-prone hosts. NC/Nga mouse-derived splenic mononuclear cells were co-cultured with NRDEP (0-50 μg/ml); thereafter, the production/release of interleukin (IL)-18 in the culture supernatants was evaluated by means of ELISA. NRDEP increased IL-18 production/release by splenocytes in a dose-dependent manner with an overall trend (with significance vs. 10 μg/ml of NRDEP). In contrast, 50 μg/ml of NRDEP inhibited production/release. These results suggest that NRDEP can activate naïve splenic mononuclear cells from atopy-prone animals in terms of IL-18 induction.
The severe incident of Fukushima Daiichi Nuclear Power Station has caused radioactive contamination of environment including drinking water. Radioactive iodine, cesium, strontium, barium and zirconium are hazardous fission products because of the high yield and/or relatively long half-life. In the present study, 4 pot-type water purifiers and several adsorbents were examined for the removal effects on these elements from drinking water. Iodide, iodate, cesium and barium were removed by all water purifiers with efficiencies about 85%, 40%, 75-90% and higher than 85%, respectively. These efficiencies lasted for 200 l, which is near the recommended limits for use of filter cartridges, without decay. Strontium was removed with initial efficiencies from 70% to 100%, but the efficiencies were slightly decreased by use. Zirconium was removed by two models, but hardly removed by the other models. Synthetic zeolite A4 efficiently removed cesium, strontium and barium, but had no effect on iodine and zirconium. Natural zeolite, mordenite, removed cesium with an efficiency as high as zeolite A4, but the removal efficiencies for strontium and barium were far less than those of zeolite A4. Activated carbon had little removal effects on these elements. In case of radioactive contamination of tap water, water purifiers may be available for convenient decontamination of drinking water in the home.
This study was designed to evaluate changes in auditory brainstem response (ABR) in the course of auditory disturbance in rats induced by Kanamycin (KM). KM was administered subcutaneously to 12 CD (SD) male rats aged 6 weeks for 10 days at a dose of 800 mg/kg. Death was observed in one male on day 8 and 2 males on day 10. It was thought that kidney damage was the cause of death from histopathological findings. ABR was recorded before KM treatment and on days 4, 8, 10 and 11 after KM treatment. The ABR changes after KM treatment in rats were as follows. On day 4, 6 rats showed an increase in amplitude of waves I and/or II and on day 8, among those, 4 rats still showed a high amplitude of waves I and/or II. On day 8, 2 rats showed an elevation of ABR threshold (15-40 dB SPL) and a decrease in amplitude of wave I and increase in amplitude of wave II at the same time. On day 11, 7 rats showed a decrease in amplitude of wave I. In addition, ABR threshold shifts (10-70 dB SPL) were observed in those rats. In ABR recording, KM-induced auditory disturbance model rats showed an increase in amplitude of waves I and/or II earlier than an ABR threshold shift. By analyzing temporal alteration of amplitude of the ABR components, we could detect precursory phenomenon of the auditory disturbance at an early phase of treatment. By following the pathway of click-ABR and tone pip-ABR examination, the auditory disturbance of low- frequency to high-frequency range could be analyzed at an early date in detail.
To investigate the adaptive response to the environmental electrophile methylmercury (MeHg), we performed DNA microarray analysis of human neuroblastoma SH-SY5Y cells exposed to a sub-cytotoxic dose of MeHg (1 µM) for 6 hr. The expression of 15 genes increased 10-fold or more in response to MeHg. Four of these genes are associated with detoxification and excretion of MeHg into the extracellular space, and are regulated by transcription factor Nrf2 through the electrophile response element. Interestingly, Cullin3, a negative regulator of Nrf2, was identified as a down-regulated gene during MeHg exposure.
Cadmium is a typical environmental pollutant and the causative agent of itai-itai disease. Ubiquitin ligases potentially involved in mediating the effects of cadmium toxicity were investigated by RNA interference using a panel of small inhibitory (si)RNAs. The results demonstrated that the inhibition of gene expression of FBXO6 sensitizes HEK293 cells to cadmium.
Methylmercury is a hazardous heavy metal compound that can damage the central nervous system. The mechanisms of methylmercury toxicity and the biological defense mechanisms against it remain unclear. We employed gene knockdown using siRNA to search for transcription factors involved in the manifestation of methylmercury toxicity. We found that the inhibition of expression of the gene for heat shock transcription factor 1 (HSF1) sensitized HEK293 cells to methylmercury.
To elucidate the mechanisms underlying adriamycin resistance, adriamycin resistance-related genes were explored using the budding yeast Saccharomyces cerevisiae as a useful eukaryotic model. The CLN1 and CLN2 genes, encoding G1 cyclin, and the ERG13 gene, encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, were identified.
Genes that are essential for growth in yeast were screened to identify those involved in arsenite sensitivity. We found that the knockdown of YPT1, ERG8, or RKI1 enhanced arsenite sensitivity in yeast.