Effect of glutathione depletion, hyperthermia, and a 100-mT static magnetic field on an hsp70/luc reporter system.

Heat shock proteins, in particular hsp70, are induced under conditions of cellular stress. It has been reported that environmental stimuli such as hyperthermia, oxidative stress, and exposure to magnetic fields increase levels of hsp70. It has also been reported that hyperthermia in combination with magnetic field exposure results in a synergistic increase in hsp70 production. We tested the hypothesis that oxidative stress induced by glutathione (GSH) depletion in combination with static magnetic field (SMF) exposure will produce a similar synergistic increase in hsp70 production. We exposed cells to heat, SMF, and diethylmaleate (DEM), which depletes GSH levels alone and in combination with each other, and measured hsp70 production using an hsp70/luciferase reporter and mRNA levels using PCR. We found that treatment with DEM significantly reduced the rate of luciferase bioluminescence production, particularly in the presence of heat. There was no significant effect of a 100-mT SMF exposure either alone or in combination with heat, DEM, or both on bioluminescence, however there was a significant interaction between SMF and DEM on hsp70 mRNA levels. Therefore, under our exposure conditions, GSH depletion reduced hsp70 levels but a synergistic effect of combining this stress with other external stimuli was only observed at the level of mRNA.

Localization of spherical lesions in tumor-mimicking phantoms by 3D sparse array photoacoustic imaging.

PURPOSE: The authors have developed a sparse-array photoacoustic imaging (SPAI) system that is capable of mapping 3D distributions of optical absorption using a small number of laser pulses with no mechanical scanning needed. In previous studies, the authors have shown the localization accuracy and the high frame-rate image acquisition on simple phantoms with limited medical relevance. The purpose of this study was to test the imaging capabilities of SPAI in the context of breast tumor detection and localization. METHODS: The authors constructed an array of phantoms that include spherical lesions of sizes 1.5-9 mm, buried in highly scattering tissue phantoms at depths of 3-30 mm. The authors investigated both homogeneous lesions made of blood at varying concentrations and heterogeneous lesions containing vessel-like structures. Volumetric images of the deeply buried lesions were taken at increasingly shallower depths and image-based localization was compared to measured depth. RESULTS: The authors were able to detect and accurately localize homogeneous lesions having a realistic absorption coefficient of 0.2 cm(-1) down to depths of 9-20 mm, and heterogeneous lesions containing 0.5 mm diameter vessel-like structures down to depths of 13-20 mm. Image acquisition required 2.5 s for each volumetric lesion image. CONCLUSIONS: These results suggest that 3D SPAI can detect highly vascularized lesions well below 1 cm in diameter and can overcome optical scatter of tissue to depths of 1-2 cm. With further improvement in the sensitivity and noise characteristics of the imaging system, similar imaging depths should be within reach in real breast tissue. The method, due to its optical contrast, 3D imaging, and fast acquisition, may prove useful in the clinic as an adjunct to existing breast screening tools.

Angular domain fluorescence imaging for small animal research.

We describe a novel macroscopic fluorescent imaging technique called angular domain fluorescence imaging (ADFI) applicable to the detection of fluorophores embedded in biological tissues. The method exploits the collimation detection capabilities of an angular filter array (AFA). The AFA uses the principle of acceptance angle filtration to extract minimally scattered photons emitted from fluorophores deep within tissue. Our goal was to develop an ADFI system for imaging near-infrared fluorescent markers for small animal imaging. According to the experimental results, the ADFI system offered higher resolution and contrast compared to a conventional lens and lens-pinhole fluorescent detection system. Furthermore, ADFI of a hairless mouse injected with a fluorescent bone marker revealed vertebral structural and morphometric data that correlated well with data derived from volumetric x-ray computed tomography images. The results suggested that ADFI is a useful technique for submillimeter mapping of the distribution of fluorescent biomarkers in small animals.

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system.

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real-time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real-time. Two heat parameters were considered in combination with sham- and 100 mT-exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult.

Effect of 100 mT homogeneous static magnetic field on [Ca2+]c response to ATP in HL-60 cells following GSH depletion.

Calcium is an important molecule in a number of biological systems. Often these systems are signal transduction cascades involving molecules such as ATP. ATP activates second messengers which can interact with ion channels on the endoplasmic/sarcoplasmic reticulum resulting in the emptying of the intracellular calcium stores and an increase in cytosolic free calcium concentration ([Ca2+]c). Changes in [Ca2+]c can be influenced by external factors such as a static magnetic field (SMF). One hypothesis suggests that a SMF affects the cells through the radical pair mechanism. By reducing the number of antioxidant molecules like glutathione (GSH), the proportion of free radicals in the cells is increased and may lead to a greater probability of a biological response to a SMF. The purpose of this study was to determine if the [Ca2+]c response to ATP was affected by depletion of GSH by diethylmaleate (DEM) and the absence or presence of a 100 mT homogeneous SMF. Undifferentiated HL-60 cells were loaded with fura-2 AM. [Ca2+]c was measured in real time using a ratiometric fluorescence spectroscopy system. Various (DEM) ranging from 1 to 15 mM were added to deplete GSH. Cells were either exposed to sham or magnetic field (100 mT) for 13 min (780 s) and challenged with 1 microM ATP. The data show that [Ca2+]c was elevated following treatment with DEM with greater [Ca2+]c at higher [DEM]. The [Ca2+]c response to ATP was decreased as the DEM concentration increased. However, there was no effect of a 100 mT SMF on the average [Ca2+]c peak following ATP activation or the full width at half maximum (FWHM) of the [Ca2+]c response and recovery after ATP activation.

Real-time measurement of cytosolic free calcium concentration in DEM-treated HL-60 cells during static magnetic field exposure and activation by ATP.

This study investigated whether glutathione depletion affected the sensitivity of HL-60 cells to static magnetic fields. The effect of Diethylmaleate (DEM) on static magnetic field induced changes in cytosolic free calcium concentration ([Ca(2+)](c)) was examined. Cells were loaded with a fluorescent dye and exposed to a uniform static magnetic field at a strength of 0 mT (sham) or 100 mT. [Ca(2+)](c) was monitored during field and sham exposure using a ratiometric fluorescence spectroscopy system. Cells were activated by the addition of ATP. Metrics extracted from the [Ca(2+)](c) time series included: average [Ca(2+)](c) during the Pre-Field and Field Conditions, peak [Ca(2+)](c) following ATP activation and the full width at half maximum (FWHM) of the peak ATP response. Comparison of each calcium metric between the sham and 100 mT experiments revealed the following results: average [Ca(2+)](c) measured during the Field condition was 53 +/- 2 nM and 58 +/- 2 nM for sham and 100 mT groups, respectively. Average FWHM was 51 +/- 3 s and 54 +/- 3 s for sham and 100 mT groups, respectively. An effect of experimental order on the peak [Ca(2+)](c) response to ATP in sham/sham experiments complicated the statistical analysis and did not allow pooling of the first and second order experiments. No statistically significant difference between the sham and 100 mT groups was observed for any of the calcium metrics. These data suggested that manipulation of free radical buffering capacity in HL-60 cells did not affect the sensitivity of the cells to a 100 mT static magnetic field.

Real-time measurement of cytosolic free calcium concentration in HL-60 cells during static magnetic field exposure and activation by ATP.

Calcium ions are involved in a number of important signal transduction pathways in cells. Cytosolic calcium concentration ([Ca(2+)](c)) can be affected by the activation of Ca(2+) channels through the action of ligands such as ATP. The response of [Ca(2+)](c) to ligands may be affected by external factors like magnetic fields. The purpose of this study was to determine if exposure to a static magnetic field (SMF) for 800 s altered the [Ca(2+)](c) response to ATP in undifferentiated HL-60 cells. We sham exposed or field exposed fura-2 loaded HL-60 cells to a SMF of 1, 10, and 100 mT. Cells were activated with ATP 300 s into the exposure. The level of [Ca(2+)](c) was followed before, during, and after field or sham exposure with a ratiometric fluorescence spectroscopy system. It was found that high concentrations of ATP resulted in greater [Ca(2+)](c) responses, but faster recovery to near basal levels. The application of 1, 10, or 100 mT SMF did not affect the [Ca(2+)](c) response to ATP. Future work could examine the effect of a longer SMF exposure on the [Ca(2+)](c) response to ATP. Longer exposures might provide sufficient time for morphological changes in the plasma membrane to occur.