Carl F. Blackman

Effects of ELF Fields on Calcium-Ion Efflux from Brain Tissue in Vitro

It has been previously demonstrated that carrier waves of 50 and 147 MHz, when sinusoidally amplitude modulated at 16 Hz (ELF), can cause enhanced efflux of radiolabeled calcium ions from chick brain tissue in vitro. This phenomenon occurs only when the samples are exposed to specific intensity ranges of the carrier wave. Unmodulated carrier waves do not affect the ion efflux. Since the ELF signal must be demodulated from the carrier wave to be effective, a study of the efflux ehnancement due to the ELF signal alone may lead to an identification of the site of demodulation, as well as provide clues to the underlying mechanism. We report here that 16-Hz sinusoidal fields in the absence of a carrier wave can alter the efflux rate of calcium ions. The results show a frequency-dependent, field-induced enhancement of calcium-ion efflux within the ranges 5 to 7.5 V/m and 35 to 50 V/m (peak-to-peak incident field in air) with no enhancement within the ranges 1 to 2, 10 to 30, and 60 to 70 V/m.

Effect of treatment media on the agglomeration of titanium dioxide nanoparticles: impact on genotoxicity, cellular interaction, and cell cycle.

The widespread use of titanium dioxide (TiO2) nanoparticles in consumer products increases the probability of exposure to humans and the environment. Although TiO2 nanoparticles have been shown to induce DNA damage (comet assay) and chromosome damage (micronucleus assay, MN) in vitro, no study has systematically assessed the influence of medium composition on the physicochemical characteristics and genotoxicity of TiO2 nanoparticles. We assessed TiO2 nanoparticle agglomeration, cellular interaction, induction of genotoxicity, and influence on cell cycle in human lung epithelial cells using three different nanoparticle-treatment media: keratinocyte growth medium (KGM) plus 0.1% bovine serum albumin (KB); a synthetic broncheoalveolar lavage fluid containing PBS, 0.6% bovine serum albumin and 0.001% surfactant (DM); or KGM with 10% fetal bovine serum (KF). The comet assay showed that TiO2 nanoparticles induced similar amounts of DNA damage in all three media, independent of the amount of agglomeration, cellular interaction, or cell-cycle changes measured by flow cytometry. In contrast, TiO2 nanoparticles induced MN only in KF, which is the medium that facilitated the lowest amount of agglomeration, the greatest amount of nanoparticle cellular interaction, and the highest population of cells accumulating in S phase. These results with TiO2 nanoparticles in KF demonstrate an association between medium composition, particle uptake, and nanoparticle interaction with cells, leading to chromosomal damage as measured by the MN assay.

Evidence for direct effect of magnetic fields on neurite outgrowth.

Electric fields can cause changes in cell responses both in vitro and in vivo. Alternating magnetic fields have been proposed to act through the electric fields induced in the conducting medium surrounding the cells. We have used a simple exposure system to test the relative contribution of magnetic fields compared to induced electric fields in a standard PC‐12 cell culture assay, in which cells respond to nerve growth factor by producing neurites. This response to stimulation by nerve growth factor is inhibited by sinusoidal, 50‐Hz magnetic fields at field strengths below 10 μT (100 mG). A standard procedure to distinguish magnetic‐ vs. electric‐field effects demonstrates that the induced electric field is not involved. Additional work is necessary to identify the critical reaction site (or sites), and to establish the molecular mechanisms responsible for these results.—Blackman, C. F., Benane, S. G., House, D. E. Evidence for direct effect of magnetic fields on neurite outgrowth. FASEB J. 7: 801‐806; 1993.

The influence of 1.2 ?T, 60 Hz magnetic fields on melatonin- and tamoxifen-induced inhibition of MCF-7 cell growth

We independently examined the findings of Harland and Liburdy, who reported that 1.2 microT(rms), 60 Hz magnetic fields could significantly reduce the inhibitory action of physiological levels of melatonin (10(-9) M) and of pharmacological levels of tamoxifen (10(-7) M) on the growth of MCF-7 human breast cancer cells in vitro. We used two testing protocols. In the melatonin study, the cell numbers per dish on day 7 of treatment were determined using a hemocytometer assay. In the tamoxifen study we used an expanded protocol, employing an alternative cell counting assay to characterize the cell numbers per dish on days 4, 5, 6, and 7. In both the melatonin and tamoxifen studies, cells were plated on 35 mm dishes and placed in each of two exposure chambers inside 5% CO(2) incubators. One exposure chamber was energized to produce 1.2 microT(rms), 60 Hz magnetic fields and the other chamber was not energized. Treatment was continuous until assays were performed. Cells were harvested at selected times, and enumerated without knowledge of treatment. In the melatonin study, the experiment was repeated three times, whereas in the tamoxifen study, each experiment was repeated nine times. In the melatonin study, cell numbers per dish were significantly reduced (by 16.7%) in the melatonin treated cultures after 7 days of incubation compared to control cultures, whereas in the presence of 1.2 microT(rms), 60 Hz magnetic fields, the melatonin treated cultures had the same cell populations as the control cultures. In the tamoxifen study, tamoxifen reduced the cell growth by 18.6 and 25% on days 6 and 7, respectively, in the chamber not energized, while in 1.2 microT(rms), 60 Hz fields, tamoxifen reduced the cell growth only by 8.7 and 13.1%, respectively. These results are consistent with those reported by Harland and Liburdy. A critical element of this successful replication effort was the constructive communication established and maintained with the original investigators. Bioelectromagnetics 22:122-128, 2001. Published 2001 Wiley-Liss, Inc.

The ion parametric resonance model predicts magnetic field parameters that affect nerve cells.

An ion parametric resonance (IPR) model recently developed by Blanchard and Blackman predicts distinct magnetic field interactions with biological systems based on a selective relation among four factors: the flux density of the static magnetic field, the frequency and flux density (Bac) of the parallel ac magnetic field, and the charge‐to‐mass ratio of ions of biological relevance. To test this model, PC‐12 cells stimulated by nerve growth factor to produce neurites were exposed for 23 h in a 5% CO2 incubator using a multiple‐coil exposure system to produce 45 Hz ac and dc (366 mG parallel to ac; less than 2 mG perpendicular to ac) magnetic fields. Our earlier work showed a cycle of inhibition/no inhibition of neurite outgrowth consistent with the IPR model predictions for Bac exposures between 0 and 468 mG rms. The work described here tests neurite outgrowth over a broader range of Bac (233–1416 mG rms). The experimental results remain consistent with earlier results, and with IPR model predictions of a second cycle of inhibition, return to control values, followed by a third cycle of inhibition of neurite outgrowth. These responses support the fundamental relationships predicted by the IPR model. The results have broad significance for biology.—Blackman, C. F., Blanchard, J. P., Benane, S. G., House, D. E. The ionic parametric resonance model predicts magnetic Field parameters that affect nerve cells. FASEB J. 9, 547–551 (1995)

Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: Effect of size, surface coating, and intracellular uptake ☆

Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key stress-responsive pathways activated by Nrf-2/ARE, NFκB, and AP1 during exposure to Ag NP of two distinct sizes (10 and 75 nm) and coatings (citrate and polyvinylpyrrolidone), as well as silver nitrate (AgNO3), and CeO2 nanoparticles. The in vitro assays assessed the cellular response in a battery of stable luciferase-reporter HepG2 cell lines. We further assessed the impact of Ag NP and AgNO3 exposure on cellular redox status by measuring glutathione depletion. Lastly, we determined intracellular Ag concentration by inductively coupled plasma mass spectroscopy (ICP-MS) and re-analyzed reporter-gene data using these values to estimate the relative potencies of the Ag NPs and AgNO3. Our results show activation of all three stress response pathways, with Nrf-2/ARE displaying the strongest response elicited by each Ag NP and AgNO3 evaluated here. The smaller (10-nm) Ag NPs were more potent than the larger (75-nm) Ag NPs in each stress-response pathway, and citrate-coated Ag NPs had higher intracellular silver concentrations compared with both PVP-coated Ag NP and AgNO3. The cellular stress response profiles after Ag NP exposure were similar to that of AgNO3, suggesting that the oxidative stress and inflammatory effects of Ag NP are likely due to the cytotoxicity of silver ions.

Magnetic fields at resonant conditions for the hydrogen ion affect neurite outgrowth in PC-12 cells: A test of the ion parametric resonance model

PC-12 cells primed with nerve growth factor (NGF) were exposed to sinusoidal extremely-low-frequency (ELF) magnetic fields (MFs) selected to test the predictions of the ion parametric resonance (IPR) model under resonance conditions for a single ion (hydrogen). We examined the field effects on the neurite outgrowth (NO) induced by NGF using three different combinations of flux densities of the parallel components of the AC MF (Bac) and the static MF (Bdc). The first test examined the NO response in cells exposed to 45 Hz at a Bdc of 2.96 microT with resonant conditions for H+ according to the model. The Bac values ranged from 0.29 to 4.11 microT root-mean-square (rms). In the second test, the MF effects at off-resonance conditions (i.e., no biologically significant ion at resonance) were examined using the frequency of 45 Hz with a Bdc of 1.97 microT and covering a Bac range between 0.79 and 2.05 microT rms. In the third test, the AC frequency was changed to 30 Hz with the subsequent change in Bdc to 1.97 microT to tune for H+ as in the first test. The Bac values ranged from 0.79 to 2.05 microT rms. After a 23 h incubation and exposure to the MF in the presence of NGF (5 ng/ml), the NO was analyzed using a stereoscopic microscope. The results showed that the NGF stimulation of neurite outgrowth (NSNO) was affected by MF combinations over most of the Bac exposure range generally consistent with the predictions of the IPR model. However, for a distinct range of Bac where the IPR model predicted maximal ionic influence, the observed pattern of NSNO contrasted sharply with those predictions. The symmetry of this response suggests that values of Bac within this distinct range may trigger alternate or additional cellular mechanisms that lead to an apparent lack of response to the MF stimulus.

Melatonin enhances junctional transfer in normal C3H/10T1/2 cells☆

Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. The pineal gland and its hormone, melatonin, are believed to intervene in the control of neoplastic processes. Several possible mechanisms have been suggested to be potentially responsible for melatonins oncostatic action; however, the actual mechanisms involved in melatonins effects at the cellular level remain unidentified. In the present study low-density cultures of C3H/10T1/2 mouse embryo fibroblasts were incubated until relatively quiescent monolayers were established (17-18 days). Gap junctional intercellular communication in control samples and in cells treated with 10(-12) to 10(-8) M melatonin was determined by the scrape-loading assay using the fluorescent dye Lucifer yellow. The results showed that concentrations of melatonin considered physiological (10(-11) and 10(-10) M) induced a significant increase in the transfer of the dye to adjacent cells through gap junctions; both higher and lower concentrations were ineffective. These results suggest that melatonin could exert its putative oncostatic action, in part, by modulating the levels of gap junctional intercellular communication.

An increase in glial fibrillary acidic protein follows brain hyperthermia in rats.

Previously, we have demonstrated that an increase in the astrocyte-associated protein, glial fibrillary acidic protein (GFAP), accompanies brain injury induced by a variety of chemical insults. In the present study we examined the effects of microwave-induced hyperthermia of the CNS on the concentration of GFAP in several brain regions of the Long-Evans rat. Irradiation resulted in a time-related increase in GFAP in olfactory bulbs and cortex, areas of maximum heating. The increase in GFAP following a brain temperature increase suggests that heating of brain tissue may be sufficient to provoke an injury response comparable to that induced by chemical and physical insult.

A potential microRNA signature for tumorigenic conazoles in mouse liver.

Triadimefon, propiconazole, and myclobutanil are conazoles, an important class of agricultural fungicides. Triadimefon and propiconazole are mouse liver tumorigens, while myclobutanil is not. As part of a coordinated study to understand the molecular determinants of conazole tumorigenicity, we analyzed the microRNA expression levels in control and conazole‐treated mice after 90 d of administration in feed. MicroRNAs (miRNAs) are small noncoding RNAs composed of approximately 19–24 nucleotides in length, and have been shown to interact with mRNA (usually 3′ UTR) to suppress its expression. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Groups of mice were fed either control diet or diet containing 1800 ppm triadimefon, 2500 ppm propiconazole, or 2000 ppm myclobutanil. MicroRNA was isolated from livers and analyzed using Superarray whole mouse genome miRNA PCR arrays from SABioscience. Data were analyzed using the significance analysis of microarrays (SAM) procedure. We identified those miRNAs whose expression was either increased or decreased relative to untreated controls with q ≤ 0.01. The tumorigenic conazoles induced many more changes in miRNA expression than the nontumorigenic conazole. A group of 19 miRNAs was identified whose expression was significantly altered in both triadimefon‐ and propiconazole‐treated animals but not in myclobutanil‐treated animals. All but one of the altered miRNAs were downregulated compared to controls. This pattern of altered miRNA expression may represent a signature for tumorigenic conazole exposure in mouse liver after 90 d of treatment. Published 2010 Wiley‐Liss, Inc.