Aggregation is a critical cause of poor transfer into the brain tissue of intravenously administered cationic PAMAM dendrimer nanoparticles.

Dendrimers have been expected as excellent nanodevices for brain medication. An amine-terminated polyamidoamine dendrimer (PD), an unmodified plain type of PD, has the obvious disadvantage of cytotoxicity, but still serves as an attractive molecule because it easily adheres to the cell surface, facilitating easy cellular uptake. Single-photon emission computed tomographic imaging of a mouse following intravenous injection of a radiolabeled PD failed to reveal any signal in the intracranial region. Furthermore, examination of the permeability of PD particles across the blood-brain barrier (BBB) in vitro using a commercially available kit revealed poor permeability of the nanoparticles, which was suppressed by an inhibitor of caveolae-mediated endocytosis, but not by an inhibitor of macropinocytosis. Physicochemical analysis of the PD revealed that cationic PDs are likely to aggregate promptly upon mixing with body fluids and that this prompt aggregation is probably driven by non-Derjaguin-Landau- Verwey-Overbeek attractive forces originating from the surrounding divalent ions. Atomic force microscopy observation of a freshly cleaved mica plate soaked in dendrimer suspension (culture media) confirmed prompt aggregation. Our study revealed poor transfer of intravenously administered cationic PDs into the intracranial nervous tissue, and the results of our analysis suggested that this was largely attributable to the reduced BBB permeability arising from the propensity of the particles to promptly aggregate upon mixing with body fluids.

Effects of PAMAM dendrimers with various surface functional groups and multiple generations on cytotoxicity and neuronal differentiation using human neural progenitor cells.

Polyamidoamine (PAMAM) dendrimers have potential for biological applications as delivery systems for genes, drugs, and imaging agents into the brain, but their developmental neurotoxicity remains unknown. We investigated the effects of PAMAM dendrimers with various surface functional groups and multiple generations on neuronal differentiation using human neural progenitor cells at an equal mass concentration. Only PAMAM dendrimers containing amine (NH2) surface groups at concentrations of 10 µg/mL significantly reduced cell viability and neuronal differentiation, compared with non-amine-terminated dendrimers. PAMAM-NH2 with generation (G)3, G4, G5 G6, and G7 significantly decreased cell viability and inhibited neuronal differentiation from a concentration of 5 µg/mL, but G0, G1, and G2 dendrimers did not have any effect at this concentration. Cytotoxicity indices of PAMAM-NH2 dendrimers at 10 µg/mL correlated well with the zeta potentials of the particles. Surface group density and particle number in unit volume is more important characteristic than particle size to influence cytotoxicity for positive changed dendrimers. PAMAM-50% C12 at 1 µg/mL altered the expression level of the oxidative stress-related genes, ROR1, CYP26A1, and TGFB1, which is a DNA damage response gene. Our results indicate that PAMAM dendrimer exposure may have a surface charge-dependent adverse effect on neuronal differentiation, and that the effect may be associated with oxidative stress and DNA damage during development of neural cells.

Effects of Polyamidoamine Dendrimers on a 3-D Neurosphere System Using Human Neural Progenitor Cells.

The practical application of engineered nanomaterials or nanoparticles like polyamidoamine (PAMAM) dendrimers has been promoted in medical devices or industrial uses. The safety of PAMAM dendrimers needs to be assessed when used as a drug carrier to treat brain disease. However, the effects of PAMAM on the human nervous system remain unknown. In this study, human neural progenitor cells cultured as a 3D neurosphere model were used to study the effects of PAMAM dendrimers on the nervous system. Neurospheres were exposed to different G4-PAMAM dendrimers for 72 h at concentrations of 0.3, 1, 3, and 10 µg/ml. The biodistribution was investigated using fluorescence-labeled PAMAM dendrimers, and gene expression was evaluated using microarray analysis followed by pathway and network analysis. Results showed that PAMAM dendrimer nanoparticles can penetrate into neurospheres via superficial cells on them. PAMAM-NH2 but not PAMAM-SC can inhibit neurosphere growth. A reduced number of MAP2-positive cells in flare regions were inhibited after 10 days of differentiation, indicating an inhibitory effect of PAMAM-NH2 on cell proliferation and neuronal migration. A microarray assay showed 32 dendrimer toxicity-related genes, with network analysis showing 3 independent networks of the selected gene targets. Inducible immediate early gene early growth response gene 1 (Egr1), insulin-like growth factor-binding protein 3 (IGFBP3), tissue factor pathway inhibitor (TFPI2), and adrenomedullin (ADM) were the key genes in each network, and the expression of these genes was significantly down regulated. These findings suggest that exposure of neurospheres to PAMAM-NH2 dendrimers affects cell proliferation and migration through pathways regulated by Egr1, IGFBP3, TFPI2, and ADM.

Repulsive Apoptosis During Exposure of Mesencephalic Neural Stem Cells to Silver Nanoparticles in a Neurosphere Assay In Vitro.

Neurodevelopmental toxicity of silver nanoparticles (AgNPs) remains largely unknown. In this study, we applied a neurosphere assay for neurodevelopmental effects of AgNPs. The neural stem cells were isolated from rat mesencephalon. They were cultured as a sphere. In an assay with coated plates, cells appeared by anchoraging on the dish and then started to migrate along the radial axis from the neurosphere. AgNPs inhibited cell migration in a dose-dependent manner. There was a linear correlation between the inhibition of migration and the logarithm of the particle concentration (1.25-10 µg/ml); the half-maximal inhibitory concentration was 0.41 µg/ml for 16-h exposure. Preceding migrated cells were retarded and/or collapsed by exposure to AgNPs: lower doses of AgNPs (0.31-1.2 µg/ml) caused a 42% retardation for 48 h, while higher doses of AgNPs (2.5-10 µg/ml) collasped migrating cells. Furthermore, collapsed cells were TUNEL-positive and showed a defect in the mitochondrial membrane potential. Thus, we showed the neurodevelopmental toxicity of AgNPs using an in vitro neurosphere assay system.

Effects of PAMAM dendrimers in the mouse brain after a single intranasal instillation.

Dendrimers are highly branched spherical nanomaterials produced for use in diagnostic and therapeutic applications such as a drug delivery system. The toxicological profiles of dendrimers are largely unknown. We investigated the in vivo effects of nasal exposure to polyamidoamine (PAMAM) dendrimers on their effects on neurological biomarkers in the mouse brain. A single dose of PAMAM dendrimers (3 or 15µg/mouse) was intranasally administered to 8-week old male BALB/c mice. Twenty-four hours after administration, the olfactory bulb, hippocampus, and cerebral cortex were collected and potential biomarkers in the blood and brain were examined using blood marker, microarray and real-time RT-PCR analyses. No remarkable changes in standard serum biochemical markers were observed in the blood. A microarray analysis showed the alterations of the genes expression level related to pluripotent network, serotonin-anxiety pathway, TGF-beta receptor signaling, prostaglandin synthesis-regulation, complement-coagulation cascades, and chemokine-signaling pathway and non-odorant GPCR signaling pathways in brain tissues. Brain derived-neurotrophic factor mRNA was up-regulated in the hippocampus and cerebral cortex in mice treated with a high dose of dendrimers. These findings suggest that PAMAM dendrimers may reach the brain via the systemic circulation or an olfactory nerve route after intranasal instillation, and indicate that a single intranasal administration of PAMAM dendrimers may potentially lead to neuronal effects by modulating the gene expression of brain-derived neurotrophic factor signaling pathway.

Power-frequency magnetic fields and childhood brain tumors: a case-control study in Japan.

BACKGROUND: The strength of the association between brain tumors in children and residential power-frequency magnetic fields (MF) has varied in previous studies, which may be due in part to possible misclassification of MF exposure. This study aimed to examine this association in Japan by improving measurement techniques, and by extending measurement to a whole week. METHODS: This population-based case-control study encompassed 54% of Japanese children under 15 years of age. After excluding ineligible targeted children, 55 newly diagnosed brain tumor cases and 99 sex-, age-, and residential area-matched controls were included in the analyses. The MF exposures of each set of matching cases and controls were measured in close temporal proximity to control for seasonal variation; the average difference was 12.4 days. The mean interval between diagnosis and MF measurements was 1.1 years. The weekly mean MF level was defined as the exposure. The association was evaluated using conditional logistic regression analysis that controlled for possible confounding factors. RESULTS: The odds ratios (95% CI) for exposure categories of 0.1 to 0.2, 0.2 to 0.4, and above 0.4 microT, against a reference category of <0.1 microT, were 0.74 (0.17-3.18), 1.58 (0.25-9.83), and 10.9 (1.05-113), respectively, after adjusting for maternal education. This dose-response pattern was stable when other variables were included in the model as possible confounding factors. CONCLUSIONS: A positive association was found between high-level exposure-above 0.4 microT-and the risk of brain tumors. This association could not be explained solely by confounding factors or selection bias.

Learning behavior in rat offspring after in utero and lactational exposure to either TCDD or PCB126.

OBJECTIVES: We studied and compared the possible effects of in utero and lactational exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) or 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB126) on learning behavior in offspring. METHODS: Pregnant Long-Evans Hooded rats were administered either TCDD (50, 200, or 800 ng/kg) or PCB126 (500, 2,000 or 8,000 ng/kg) on gestational day 15. A procedure of schedule-controlled operant behavior was applied to examine learning behavior in the male and female offspring at 11 weeks of age for 30 days. Three indices, namely, response rates in a fixed ratio (FR) and in a differential reinforcement of low rates (DRL), and reward rate in the DRL component in multiple FR 20 DRL 20 s (mult-FR 20 DRL 20-s) test sessions, were used for the evaluation of learning behavior. RESULTS: Toxic effects on learning behavior in male and female pups following in utero and lactational exposure to TCDD or PCB126 were observed mainly in the FR learning component. However, no linear dose-dependent effects of either of the two compounds were observed for the above three indices. The response rates of animals in the low-dose TCDD and PCB126 groups decreased and those in medium-dose TCDD and PCB126 groups appeared to induce hyperactive behavior. The high dose of PCB126 appeared to have a distinct toxicity from that of TCDD in terms of the acquisition of learning behavior. CONCLUSIONS: Toxicities of PCB126 and TCDD in learning behavior might be similar to each other and the current toxic equivalency factor (TEF) of 0.1 for PCB126 can be considered to be appropriate for this endpoint.

Distinct requirements for interleukin-6 in airway inflammation induced by diesel exhaust in mice.

To clarify the possible role of interleukin-6 in aggravation of inflammatory responses in diesel exhaust-exposed mice, we compared the infiltration of inflammatory cells and the production of chemokines between interleukin-6-deficient and wild-type mice following 0, 1.0, or 3.0 mg diesel particles/m3 diesel exhaust inhalation for 4 weeks. Exposure to diesel exhaust significantly increased the number of inflammatory cells and the amount of CCL17 and CXCL3 in bronchoalveolar lavage fluids from wild-type mice, but not in interleukin-6-deficient mice. These findings suggest that interleukin-6 plays a critical role in airway inflammatory responses induced by diesel exhaust inhalation.

Childhood leukemia and magnetic fields in Japan: a case-control study of childhood leukemia and residential power-frequency magnetic fields in Japan.

Residential power-frequency magnetic fields (MFs) were labeled as a possible human carcinogen by the International Agency for Research on Cancer panel. In response to great public concern, the World Health Organization urged that further epidemiologic studies be conducted in high-exposure areas such as Japan. We conducted a population-based case-control study, which covered areas inhabited by 54% of Japanese children. We analyzed 312 case children (0-15 years old) newly diagnosed with acute lymphoblastic leukemia (ALL) or acute myelocytic leukemia (AML) in 1999-2001 (2.3 years) and 603 controls matched for gender, age and residential area. Weekly mean MF level was determined for the child's bedroom. MF measurements in each set of a case and controls were carried out as closely in time as possible to control for seasonal variation. We evaluated the association using conditional logistic regression models. The odds ratios for children whose bedrooms had MF levels of 0.4 microT or higher compared with the reference category (MF levels below 0.1 microT) was 2.6 (95% CI=0.76-8.6) for AML+ALL and 4.7 (1.15-19.0) for ALL only. Controlling for some possible confounding factors did not alter the results appreciably. Even an analysis in which selection bias was maximized did not fully explain the association. Most of the leukemia cases in the highest exposure category had MF levels far above 0.4 microT. Our results provided additional evidence that high MF exposure was associated with a higher risk of childhood leukemia, particularly of ALL.

Effect of diesel exhaust on immune responses in C57BL/6 mice intranasally immunized with pollen antigen.

To investigate the effect of diesel exhaust or particle-free diesel gas on immune responses in IgE low responder mice, C57BL/6 mice immunized intranasally with sugi basic protein were exposed to diesel exhaust or diesel gas components. We evaluated the changes in lymphocyte subpopulations, cell proliferation, chemokine production of cervical lymph nodes cells, and antigen-specific-antibody levels in plasma. Exposure to diesel gas decreased the percentage of CD4+ and TCR-beta+ T cells of cervical lymph nodes from immunized mice. Culture supernatants of cervical lymph nodes cells from diesel gas-exposed mice had significantly increased levels of macrophage inflammatory protein-1alpha and thymus and activation-regulated chemokine, but exposure to diesel exhaust did not affect it. Antigen-specific IgG2a titers in plasma were significantly enhanced after their exposure to diesel exhaust or gas. In contrast, exposure to diesel exhaust or gas markedly decreased antigen-specific IgG1 titers in immunized mice. These facts indicate that concurrent exposure to allergen and diesel exhaust or diesel gas modulates chemokine production in cervical lymph nodes cells and antibody production in plasma differentially.

A very low level of magnetic field exposure does not affect a participant's mental fatigue and stress as much as VDT work.

Exposure to Magnetic Fields (MF) may affect the health of workers in an occupational environment. However, there is no clear evidence that the emission of MF from the visual display terminals (VDT) affects workers'mental status or induces stress in office environments. The purpose of our study was to investigate whether exposure to the MF during VDT work affects mental fatigue or induces stress related to psycho-physiological changes in workers. Thirty-seven students were voluntarily recruited to participate in this study. We created experimental conditions that a pair of the liquid crystal display (LCD) terminals separated by screens was closely placed in front of the visual suface of the cathode ray tube (CRT) units. All participants were not able to recognize whether the power of the CRT Units was ON or OFF while they worked with the LCD terminals. When the CRT unit was ON, participants who worked with the LCD terminal were called CRT-ON group. On the other hand, when the CRT unit was OFF, participants who worked with the CLD terminal were called CRT-OFF group. Incredibly short profiles of mood states (ISP)and subjective symptom questionnaires were used as indices of the mental fatigue as well as psychological stress before and after VDT work. Furthermore, salivary chromogranin A and urinary 8-hydroxydeoxyguanosine (8-OH-dG) were collected to measure physiological changes before and after VDT work. The results of the questionnaires and salivary and urinary stress markers showed that the exposure to the MF during VDT work did not significantly induce mental fatigue or psychological stress in comparison with the CRT-OFF group. Contrary to the results from the exposure, mood states of vigor decreased greatly (P < 0.05) whereas the states of fatigue and confusion increased significantly (P < 0.001) after VDT work. In the questionnaires for subjective symptoms, participants complained remarkably regarding 14 out of 25 conditions after VDT work. Moreover, urinary 8-OH-dG excretion increased significantly after VDT work. Taken together, the results of this study did not detect any bad health effects of MF exposure on the VDT participants but suggested that VDT work itself affects their mental fatigue and psycho-physiological status.

Inhalation of low-level formaldehyde enhances nerve growth factor production in the hippocampus of mice.

OBJECTIVE: The effects of low-level formaldehyde (FA) inhalation on the amount of nerve growth factor (NGF) in the hippocampus of immunized mice were studied. METHODS: Evaluation of NGF in the hippocampus was performed by ELISA and RT-PCR. RESULTS: Exposure to 80 and 400 ppb FA significantly increased the brain NGF levels in the immunized mice. Evaluation of the NGF levels in the hippocampus of immunized mice showed that 400 ppb FA significantly increased the NGF content. The RT-PCR evaluation also showed higher concentrations of hippocampal NGF mRNA in the mice exposed to 80 and 400 ppb FA with immunization. CONCLUSION: Exposure of immunized mice to low levels of FA significantly increases NGF levels in the hippocampus.

Differential immunogenic and neurogenic inflammatory responses in an allergic mouse model exposed to low levels of formaldehyde.

It is suspected that exposure to low levels of formaldehyde induces or aggravates airway inflammation mediated by immunological and neurological reactions. To clarify the effect of this exposure on allergic inflammatory responses, we exposed female C3H/He mice to 0, 80, 400, or 2000ppb formaldehyde for 12 weeks. When mice were immunized with ovalbumin (OVA) and then exposed to formaldehyde, the numbers of total bronchoalveolar lavage cells, macrophages, and eosinophils in the mice exposed to 2000ppb formaldehyde were significantly increased compared to 0ppb controls. However, the production of interleukin-1beta from bronchoalveolar lavage fluid of these mice decreased significantly. Immunization with OVA significantly increased the production of nerve growth factor, but exposure to 80 and 400ppb formaldehyde significantly reduced the nerve growth factor levels in bronchoalveolar lavage fluid of the immunized mice. In in vitro study, markedly increased lipopolysaccharide-stimulated interferon-gamma production in culture supernatants of spleen cells from 2000ppb formaldehyde-exposed, nonimmunized mice, and significantly increased OVA-stimulated monocyte chemoattractant protein-1 production in culture supernatants of spleen cells from 400 and 2000ppb formaldehyde-exposed, immunized mice were observed. Exposure to 400ppb formaldehyde induced significant decreases in anti-OVA IgG1 and IgG3 antibody productions in plasma, whereas anti-OVA IgE antibody production was not affected. In addition, the levels of nerve growth factor in plasma of 80 and 400ppb formaldehyde-exposed, immunized mice significantly decreased compared to 0ppb control, immunized mice. These results provide the first experimental evidence that low levels of long-term formaldehyde inhalation can induce differential immunogenic and neurogenic responses in allergic mice.

Diesel exhaust-associated gas components enhance chemokine production by cervical lymph-node cells from mice immunized with sugi basic proteins.

Previously, we showed that exposure to diesel exhaust (DE) increased inflammatory cells in the airway and cytokine production from local lymph-node cells after antigen stimulation. To clarify the role of particle-free diesel gas components in induction of allergic inflammation, we compared the effect of DE and gas components on pollen-antigen-stimulated chemokine production by cervical lymph nodes (CLN) cells in BALB/c mice. Groups of mice were exposed to 0 (control), 1.0 mg diesel exhaust particles (DEP)/m(3) (DE), or filtered 1.0 mg DEP/m(3) DE (gas) for 12 h daily for 5 wk. Each group of mice was injected intraperitoneally with sugi basic protein (SBP), a major allergen of Japanese cedar pollen, immediately before their exposure to DE or gas. On days 14 and 35, each mouse received an additional SBP intranasally. Exposure to DE or gas did not affect the lymphocyte subpopulations of CLN. Culture supernatants of CLN cells from DE-exposed, SBP-immunized mice had significantly increased levels of monocyte chemoattractant protein-1. Exposure to gas significantly increased the amount of thymus- and activation-regulated chemokine and macrophage inflammatory proteins-1 alpha in the CLN cells from SBP-immunized mice. These results suggest that Gas components as well as DEP may differentially regulate production of chemokines at local sites.

Evaluation of residential exposure to intermediate frequency magnetic fields.

The authors measured the exposure to intermediate-frequency (IF: 10 kHz to 30 MHz) electromagnetic fields (EMF) in residential environments. They developed a system to acquire and record waveforms of IF magnetic fields (MFs) and set 5 nanotesla (nT) for the trigger level of acquisition. They operated the system near power lines, railroads, and electrical appliances as possible sources of IF-MFs. Most of the maximum values of magnetic flux density and the time derivative for each wave were below the upper limit of the measurable range of our system (i.e., 53 nT and 10 T/s); these values were much lower than the minimum amplitudes that can theoretically induce heating or membrane excitation within biological systems. Moreover, the amplitudes of the IF-MFs were not related to those of extremely low frequency (ELF) MF measured simultaneously, indicating that IF-MFs do not underlie the associations, observed in several epidemiological studies, between residential exposure to ELF-EMF and childhood cancer.

No influence of short-term exposure to 50-Hz magnetic fields on cognitive performance function in human.

OBJECTIVES: To examine whether 50-Hz magnetic fields acutely influence cognitive performance function in humans. METHODS: The exposure experiment was conducted in a wooden room with three-axis coils. Twenty young subjects were exposed to circularly polarized 50-Hz/20-mu T magnetic fields for 55 min, during which they were requested to perform four cognitive performance tests for evaluation of simple reaction time, time and accuracy of choice reaction, time perception, and figure perception. RESULTS: No subject detected the existence of the field. Statistical analysis revealed no significant influence of the exposure on the results of the tests. CONCLUSIONS: The results indicate that extremely low-frequency electromagnetic fields in the occupational environment are unlikely to interfere with human brain function so much as to lower working efficiency or accuracy.

Can extremely low frequency alternating magnetic fields modulate heart rate or its variability in humans?

This study is a reexamination of the possibility that exposure to extremely low frequency alternating magnetic field (ELF-MF) may influence heart rate (HR) or its variability (HRV) in humans. In a wooden room (cube with 2.7-m sides) surrounded with wire, three series of experiments were performed on 50 healthy volunteers, who were exposed to MFs at frequencies ranging from 50 to 1000 Hz and with flux densities ranging from 20 to 100 microT for periods ranging from 2 min to 12 h. In each experiment, six indices of HR/HRV were calculated from the RR intervals (RRIs): average RRI, standard deviation of RRIs, power spectral components in three frequency ranges (pVLF, pLF and pHF), and the ratio of pLF to pHF. Statistical analyses of results revealed no significant effect of ELF-MFs in any of the experiments, and suggested that the ELF-MF to which humans are exposed in their daily lives has no acute influence on the activity of the cardiovascular autonomic nervous system (ANS) that modulates the heart rate.

Acute exposure to 50 Hz magnetic fields with harmonics and transient components: lack of effects on nighttime hormonal secretion in men.

The purpose of this study was to examine whether low frequency magnetic field (MF) influences nighttime secretion of hormones, particularly melatonin. Ten healthy males stayed in the experimental room (2.7 m cube with 3 axis Merritt coils) on two separate nights. On one night, subjects were exposed to linearly polarized 50 Hz, 20 microT sinusoidal MF with the third (30%) and the fifth (10%) harmonics and repetitive transient waves (1 burst/s of 1 kHz waves, exponentially attenuated with a duration of 50 ms; initially 100 microT peak), and the other night was for blind control. During the nights (2000-0800 h, including sleeping time, 2300-0700 h), blood samples were collected from the subjects at 1 h intervals for determining the levels of plasma hormones (melatonin, growth hormone (GH), cortisol, prolactin) and at 10 min intervals from 2200 to 0200 h for observing the GH surge induced by sleep. Statistical analyses revealed no significant difference between the 2 nights in the profiles of the four hormones, and the result suggested that extremely low frequency (ELF) or intermediate frequency (IF) MF to which humans are exposed residentially has no acute effect on nighttime secretion of hormones, particularly melatonin.