Evaluation of Glucose Uptake in Normal and Cancer Cell Lines by Positron Emission Tomography.

To date, there is no definitive demonstration of the utility of positron emission tomography (PET) in studying glucose metabolism in cultured cell lines. Thus, this study was designed to compare PET to more standardized methods for the quantitative assessment of glucose uptake in nontransformed and transformed living cells and to validate PET for metabolic studies in vitro. Human colon and breast carcinoma cell lines and mouse embryo fibroblasts were evaluated for [(18)F]fluorodeoxyglucose ([(18)F]FDG) uptake by PET and autoradiography and 2-deoxyglucose (2-DG) incorporation by colorimetric assay and analyzed for the radiotoxic effects of [(18)F]FDG and the expression levels of glucose transporters. Indeed, [(18)F]FDG incorporation on PET was comparable to [(18)F]FDG uptake by autoradiography and 2-DG incorporation by colorimetric assay, although radiotracer-based methods exhibited more pronounced differences between individual cell lines. As expected, these data correlated with glucose transporters 1 to 4 and hexokinase II expression in tumor cell lines and mouse fibroblasts. Notably, [(18)F]FDG incorporation resulted in low apoptotic rates, with fibroblasts being slightly more sensitive to radiotracer-induced cell death. The quantitative analysis of [(18)F]FDG uptake in living cells by PET represents a valuable and reproducible method to study tumor cell metabolism in vitro, being representative of the differences in the molecular profile of normal and tumor cell lines.

Acute toxic effects of sonodynamic therapy on hypertrophic scar fibroblasts of rabbit ears.

The objective of this study was to observe the acute cytotoxic effects of hematoporphyrin monomethyl ether sonodynamic therapy (HMME-SDT) on hypertrophic scar fibroblasts of rabbit ears. We first assessed the effects of different irradiation times and HMME concentrations on the survival of hypertrophic scar fibroblasts using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the optimum irradiation time and HMME concentration. The hypertrophic scar fibroblast cell suspensions of the rabbit ears were divided into four groups, the survival rates were detected using the MTT assay, and the type of cell death was detected by Annexin V/propidium iodide (PI) double staining flow cytometry. Our results showed that HMME-SDT significantly reduced the viability of hypertrophic scar fibroblasts of rabbit ears at ultrasonic irradiation times of 30, 60, and 90 s, but not 10 s (P < 0.05). HMME alone had no significant effect on the cell survival rate at any irradiation time (P > 0.05). In contrast, the cell survival rate was significantly decreased at an irradiation time of 10 s and HMME concentrations of 20 and 50 µg/mL (P < 0.05). Furthermore, Annexin V/PI double staining showed necrosis and apoptosis of the hypertrophic scar fibroblasts. Given our results, HMME might be an effective sound-sensitive agent for SDT as it has a significant lethal effect on hypertrophic scar fibroblasts of rabbit ear cultured in vitro. HMME-SDT may therefore provide a new method for the treatment of hypertrophic scar formation.

In-vivo comparison of the acute retention of stem cell derivatives and fibroblasts after intramyocardial transplantation in the mouse model.

PURPOSE: Various strategies have been applied to increase the engraftment of an intramyocardial cell transplant (Tx) to treat ischemic myocardium. Thereby, co-transplanted fibroblasts (FB) improve the long-term survival of stem cell derivatives (SCD) in a murine model of myocardial infarction. For therapeutic use, the time frame in which FB exert putative supportive effects needs to be identified. Therefore, we tracked the biodistribution and retention of SCD and FB in vivo using highly sensitive positron emission tomography (PET) imaging. METHODS: Murine [(18)F]-fluorodeoxyglucose (FDG) labeled SCD and FB were transplanted after left anterior descending artery (LAD) ligation into the border zone of the ischemic area in female C57BL/6 mice. Cardiac retention and biodistribution during the initial 2 h after injection were measured via PET imaging. RESULTS: Massive initial cell loss occurred independently of the cell type. Thereby, FB were retained slightly, yet significantly better than SCD until 60 min post-injection (7.5 ± 1.7 vs. 5.2 ± 0.7% ID at 25 min and 7.0 ± 1.5 vs. 4.8 ± 0.8% ID at 60 min). Thereafter, a fraction of ∼ 5% that withstood the massive initial washout remained at the site of injection independently of the applied cell type (120 min, SCD vs. FB P = 0.64). Most of the lost cells were detected in the lungs (∼ 30 % ID). CONCLUSIONS: We were able to quantitatively define the retention and biodistribution of different cell types via PET imaging in a mouse model after intramyocardial Tx. The utmost accuracy was achieved through this cell- and organ-specific approach by correcting PET data for cellular FDG efflux. Thereby, we observed a massive initial cell loss of ∼ 95%, causing low rates of long-term engraftment for both SCD and FB. We conclude that FB are not privileged compared to SCD regarding their acute retention kinetics, and therefore exert their beneficial effects at a later time point.

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei.

Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed.

Monitoring Therapy Response of Experimental Arthritis with Radiolabeled Tracers Targeting Fibroblasts, Macrophages, or Integrin αvß3.

UNLABELLED: Rheumatoid arthritis is an autoimmune disease resulting in chronic synovial inflammation. Molecular imaging could be used to monitor therapy response, thus enabling tailored therapy regimens and enhancing therapeutic outcome. Here, we hypothesized that response to etanercept could be monitored by radionuclide imaging in arthritic mice. We tested 3 different targets, namely fibroblast activation protein (FAP), macrophages, and integrin αvß3. METHODS: Male DBA/1J mice with collagen-induced arthritis were treated with etanercept. SPECT/CT scans were acquired at 1, 24, and 48 h after injection of (111)In-RGD2 (integrin αvß3), (111)In-anti-F4/80-A3-1 (antimurine macrophage antibody), or (111)In-28H1 (anti-FAP antibody), respectively, with nonspecific controls included. Mice were dissected after the last scan, and scans were analyzed quantitatively and were correlated with macroscopic scoring. RESULTS: Experimental arthritis was imaged with (111)In-28H1 (anti-FAP), (111)In-anti-F4/80-A3-1, and (111)In-RGD2. Tracer uptake in joints correlated with arthritis score. Treatment decreased joint uptake of tracers from 23 ± 15, 8 ± 4, and 2 ± 1 percentage injected dose per gram (%ID/g) to 11 ± 11 (P < 0.001), 4 ± 4 (P < 0.001), and 1 ± 0.2 %ID/g (P < 0.01) for (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2, respectively. Arthritis-to-blood ratios (in mice with arthritis score 2 per joint) were higher for (111)In-28H1 (5.5 ± 1; excluding values > 25), (111)In-anti-F4/80-A3-1 (10.4 ± 4), and (111)In-RGD2 (7.2 ± 1) than for control (111)In-DP47GS (0.7 ± 0.5; P = 0.002), (111)In-rat IgG2b (0.5 ± 0.2; P = 0.002), or coinjection of excess RGD2 (3.5), indicating specific uptake of all tracers in arthritic joints. CONCLUSION: (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2 can be used to specifically monitor the response to therapy in experimental arthritis at the molecular level. Further studies, however, still need to be performed.

Functional bone engineering using ex vivo gene therapy and topology-optimized, biodegradable polymer composite scaffolds.

Bone tissue engineering could provide an alternative to conventional treatments for fracture nonunion, spinal fusion, joint replacement, and pathological loss of bone. However, this approach will require a biocompatible matrix to allow progenitor cell delivery and support tissue invasion. The construct must also support physiological loads as it degrades to allow the regenerated tissue to bear an increasing load. To meet these complex requirements, we have employed topology-optimized design and solid free-form fabrication to manufacture biodegradable poly(propylene fumarate)/beta-tricalcium phosphate composites. These scaffolds were seeded with primary human fibroblasts transduced with an adenovirus expressing bone morphogenetic protein-7 and implanted subcutaneously in mice. Specimens were evaluated by microcomputed tomography, compressive testing, and histological staining. New bone was localized on the scaffold surface and closely followed its designed contours. Furthermore, the total stiffness of the constructs was retained for up to 12 weeks after implantation, as scaffold degradation and tissue invasion took place.

Effects of 35 kHz, low-frequency ultrasound application in vitro on human fibroblast morphology and migration patterns.

Low-frequency ultrasound (LFU) in the frequency range 30-40 kHz administered using different delivery methods (waterbath and noncontact spray) has shown positive effects on chronic wound healing rates in humans, but the underlying mechanisms are only beginning to be explored. To examine the effects of LFU delivered at 35 kHz on the morphology and migration of human fibroblasts, real-time videography was used to record the rate and character of cultured human fibroblast migration at 8-hour increments for 48 hours in a wound assay model. Cells were treated with 35 kHz LFU or saline only (control). Cellular morphology (cell shape, packing, and apparent length) and migration patterns including orientation were analyzed using time-lapse videography. LFU delivered at a frequency of 35 kHz produced a different pattern of fibroblast migration in vitro (varied orientation versus vertical orientation for untreated cells) and altered cell morphology compared to controls. The observed pattern of migration was more varied and widely distributed across multiple angles versus the predominant parallel orientation of the migrating untreated cells. The potential implications of these findings on collagen placement in the extracellular matrix, which may affect degree of soft tissue scarring, should be further investigated.

PET imaging of cardiac wound healing using a novel [68Ga]-labeled NGR probe in rat myocardial infarction.

PURPOSE: Peptides containing the asparagine-glycine-arginine (NGR) motif bind to aminopeptidase N (CD13), which is expressed on inflammatory cells, endothelial cells, and fibroblasts. It is unclear whether radiolabeled NGR-containing tracers could be used for in vivo imaging of the early wound-healing phase after myocardial infarction (MI) using positron emission tomography (PET). PROCEDURES: Uptake of novel tracer [(68)Ga]NGR was assessed together with [(68)Ga]arginine-glycine-aspartic acid ([(68)Ga]RGD) and 2-deoxy-2-[(18) F]fluoro-D-glucose after myocardial ischemia/reperfusion (MI/R) injury using µ-PET and autoradiography, and relative expressions of CD13 and integrin ß3 were assessed in fibroblasts, inflammatory cells, and endothelial cells by immunohistochemistry. RESULTS: In the infarcted myocardium, uptake of [(68)Ga]NGR was maximal from days 3 to 7 after MI/R, and correlated with fibroblast and inflammatory cell infiltration as well as [(68)Ga]RGD uptake. CONCLUSIONS: [(68)Ga]NGR allows noninvasive and sequential determination of CD13 expression in fibroblasts and inflammatory cells by PET. This will facilitate monitoring of CD13 in the individual wound healing processes, allowing patient-specific therapies to improve outcome after MI.

Immuno-PET and Immuno-SPECT of Rheumatoid Arthritis with Radiolabeled Anti-Fibroblast Activation Protein Antibody Correlates with Severity of Arthritis.

UNLABELLED: One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA. METHODS: SPECT and PET with (111)In- and (89)Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score. RESULTS: Both (111)In-28H1 and (89)Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of (111)In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis. CONCLUSION: SPECT/CT imaging with the anti-FAP antibody (111)In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. (89)Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of (89)Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.

PCH-2 regulates Caenorhabditis elegans lifespan.

Components or downstream targets of many signaling pathways such as Insulin/IGF-1 and TOR, as well as genes involved in cellular metabolism and bioenergetics can extend worm lifespan 20% or more. The C. elegans gene pch-2 and its homologs, including TRIP13 in humans, have been studied for their functions in cell mitosis and meiosis, but have never been implicated in lifespan regulation. Here we show that over-expression of TRIP13 in human fibroblasts confers resistance to environmental stressors such as UV radiation and oxidative stress. Furthermore, pch-2 overexpression in C. elegans extends worm lifespan, and enhances worm survival in response to various stressors. Conversely, reducing pch-2 expression with RNAi shortens worm lifespan. Additional genetic epistasis analysis indicates that the molecular mechanism of pch-2 in worm longevity is tied to functions of the sirtuin family, implying that pch-2 is another chromatin regulator for worm longevity. These findings suggest a novel function of the pch-2 gene involved in lifespan determination.

The influence of microscale topography on fibroblast attachment and motility.

The ability of a cell to attach and migrate on a substrate or scaffold is important in the field of tissue engineering and biomaterials, and is thus extensively studied. When considering tissue-engineering applications, a highly porous scaffold is required to guide cell growth and proliferation in three dimensions. However existing scaffolds are less than ideal for actual applications, not only as they lack mechanical strength due to pore size and have regular distribution, but also they do not ensure cell attachment, in-growth and organisation. In this study, microfabrication technology was used to create regular arrays of pits on a two-dimensional quartz surface (7, 15 and 25 microm diameter, 20 and 40 microm spacing). The patterned surface thus exhibited spatially separated mechanical edges akin to the basic structural element of a three-dimensional network, and was used as a model system for studying the effects of substrate microgeometry on fibroblast attachment and motility. Results clearly showed that fibroblast interaction with the pit edges depended on both diameter, and therefore angle of circumference, and inter pit spacing, with the largest diameter permitting cells to enter the pits. Interestingly, the highest cell proliferation rates were recorded on the smaller pits. Such information may provide details on possible pore sizes for use in synthetic tissue engineering scaffolds that aim to support fibroblast in-growth and subsequent proliferation.

3-[123I]Iodo-L-alpha-methyl tyrosine transport into human fibroblasts and comparison with Ewing's sarcoma cells.

The cellular transport systems and the transport kinetics of [123I]IMT uptake into non-malignant extracranial cells were characterized for the first time. Human fibroblasts were chosen as non-malignant extracranial cells as they are found ubiquitous in the body. [123I]IMT is exclusively transported into fibroblasts via the sodium independent system L. An apparent Michaelis constant K(m) = 116.2 +/- 18.9 microM and a maximum transport velocity V(max) = 191.6 +/- 13.9 pmol x (10(6) cells)(-1) x min(-1) were calculated for the sodium-independent transport. These results were compared with those determined in two malignantly transformed extracranial cell lines, the human Ewing's sarcoma cell lines VH-64 and CADO-ES-1.

[18F]-2-fluoro-2-deoxyglucose transport kinetics as a function of extracellular glucose concentration in malignant glioma, fibroblast and macrophage cells in vitro.

FDG-PET is used to measure the metabolic rate of glucose. Transport and phosphorylation determine the amount of hexose analog that is phosphorylated and trapped. Competition occurs for both events, such that extracellular glucose concentration affects the FDG image. This study investigated the effect of glucose concentration on the rate of FDG accumulation in three cell lines. The results show that extracellular glucose concentration has a greater impact on the rate of FDG accumulation than the relative abundance of GLUT transporter subtypes.

Inhaled radon-induced genotoxicity in Wistar rat, Syrian hamster, and Chinese hamster deep-lung fibroblasts in vivo.

This study was performed (1) to provide a comparison of the genotoxic effects of inhaled radon and radon progeny, referred to as radon in this paper, among three species of rodents: Wistar rats, Syrian hamsters, and Chinese hamsters; (2) to determine if initial chromosome damage was related to the risk of induction of lung cancer; and (3) to evaluate the tissue repair and long-term presence of cytogenetic damage in respiratory tract cells. These species were selected because Syrian hamsters are very resistant to radon induction of lung cancer and Wistar rats are sensitive; no literature is available on the in vivo effects of radon in the Chinese hamster. Exposure-response relationships were established for the rats and Syrian hamsters while the Chinese hamsters received a single exposure of radon. At 4 h (0.2 days), 15 days, and 30 days after the highest WLM exposure to radon, Wistar rats, Chinese hamsters, and Syrian hamsters were killed, and lung fibroblasts were isolated and grown in culture to determine the frequency of induced micronuclei. Animals at each level of exposure showed an increase in the frequency of micronuclei relative to that in controls (P < 0.05). The exposure-response relationship data for rats and Syrian hamsters killed 0.2 days after the end of exposure were fit to linear equations (micronuclei/1000 binucleated cells = 15.5 +/- 14.4 + 0.53 +/- 0.06 WLM and 38.3 +/- 15.1 + 0.80 +/- 0.08 WLM, respectively). For the single exposure level used (496 WLM) in Chinese hamsters killed at 0.2 days after exposure, the frequency of micronuclei/1000 binucleated cells/WLM was 1.83 +/- 0.02. A comparison of the sensitivity for induction of micronuclei/WLM illustrated that Chinese hamsters were three times more sensitive than rats. The Syrian hamsters also showed a significantly elevated response (P < 0.05) relative to rats. These data suggest that initial chromosome damage is not the major factor responsible for the high rate of radon-induced cancer in rats relative to Syrian hamsters. The frequency of micronuclei in radon-exposed rats, Syrian hamsters, and Chinese hamsters significantly decreased (P < 0.05) as a function of time after the exposure. The rate of loss of damaged cells from the lung was greatest in the Chinese hamsters, followed by Wistar rats and Syrian hamsters, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Erosion of artificial endothelia in vitro by pulsed ultrasound: acoustic pressure, frequency, membrane orientation and microbubble contrast agent dependence.

The erosion of cells from fibroblast monolayers simulating the vascular endothelium by 20 micros pulses of ultrasound at 500 Hz PRF was studied in relation to the peak negative acoustic pressure (P-; 0.0-2.5 MPa), ultrasound (US) frequency (1.0, 2.1 or 3.5 MHz), orientation of the monolayer (i.e., simulating the sites of ultrasound entry/exit from a blood vessel) and the presence or absence of a microbubble contrast agent (3 Vol% Albunex). The a priori hypotheses were that erosion of the monolayers would: 1. arise due to insonation treatment, 2. arise as a consequence of cavitation activity and, thus, increase with increasing P- at constant frequency, and decrease with increasing frequency at constant P-, 3. be significantly increased by the presence of a microbubble contrast agent, and 4. have a weak dependence on monolayer orientation. The data support these hypotheses. Under the most severe exposure conditions used, most of the affected cells appeared to have been lysed; however, a substantial number of viable cells were dislodged from the monolayer surface.

High-frequency ultrasound properties of multicellular spheroids during heating.

High-frequency ultrasound monitoring is a possible method for real-time imaging of thermal therapy in tissues at microscopic resolution. The objective of this work was to measure changes in the ultrasound properties of V79 spheroids (grown from Chinese Hamster lung fibroblasts) exposed to heating. Spheroids are clonal aggregates of cells that provide a useful model for investigating the ultrasound properties of cells in the absence of connective tissue. Relative echo signal power and attenuation coefficients were measured over the frequency range 30 MHz to 70 MHz, from spheroids heated from 37 degrees C to 50 degrees C or 60 degrees C. Echo signal power from the viable rim decreased during the first 5 min by a factor of 1.08 before the spheroid reached 50 degrees C. For the next 25 min, echo signal power rose to a factor of 1.27 above the initial level, after which it remained relatively constant over the remainder of the 50 degrees C heating period. At 60 degrees C, echo signal from the viable rim remained relatively constant, although it appeared to have possibly decreased slightly over the duration of the heating period. Echo signal power from the necrotic core fell to a factor of 1.4 and 1.54 below the initial level at 50 degrees C and 60 degrees C, respectively. First-order chemical rate analysis applied to the echo signal power results in the viable rim at 50 degrees C revealed a rate constant for the 5-15-min heating interval. Interpretation of the echo signal power results in terms of histological stains indicates that the rise in echo signal power at 50 degrees C was due to a loss of cell cohesion, and the possible drop in echo signal power at 60 degrees C was due to spheroid coagulation. Attenuation coefficients decreased by up to 1.54 dB mm-1 over a 30-min period at 60 degrees C. The appearance of a real-time ultrasound image of lesion formation in cells is discussed.

The response of normal and malignant cells to ultrasound in vitro.

The effect of ultrasonic irradiation on the viability of normal and tumor cell cultures derived from human and mouse origins was investigated. The cells were irradiated with a frequency of 2 MHz and intensity of 0.33 W/cm2, up to 4 min and immediately tested for cell viability using four different parameters: vital staining for the determination of the rate of cell growth; [3H]-thymidine and [3H]-leucine incorporation as an indication of the rate of DNA and protein synthesis respectively; and cloning efficiency as a measurement of the cell ability to multiply. Two human normal cell lines used in our studies, FS11 foreskin fibroblasts and Wish cells, were relatively resistant to ultrasonic irradiation effect although the growth rate of the latter was somewhat affected, particularly after 2 or 4 min of irradiation. However, cells derived from either malignant melanoma or breast carcinoma were highly sensitive to irradiation as demonstrated by a reduction of 96% and 65%, respectively, in cloning efficiency even after irradiation for 1 min. A third tumor cell line derived from lung carcinoma was more resistant. Two normal clones derived from NIH/3T3 mouse fibroblasts were used. These clones revealed some degree of sensitivity, particularly after 4 min of irradiation. However, their murine-sarcoma-virus transformed counterparts were found to be even more sensitive at identical times of ultrasonic irradiation, although the differences are not as striking as demonstrated with cells from human origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Sonochemicals increase the mutation frequency of V79 cells in vitro.

Phosphate buffered saline (PBS) was insonated or sham-insonated (1 MHz, 35 W/cm2, continuous wave, 30 min) in rotating (200 rpm) sterile polystyrene culture tubes. After treatment, the PBS was used immediately to suspend washed Chinese hamster V79 cells in vitro. Cells were incubated in the PBS at 37 degrees C for 15 min and then transferred to complete growth medium. Some insonation regimens also involved the inclusion of Albunex (ALX; an ultrasound microbubble contrast agent) to enhance ultrasound-induced inertial cavitation. Following exposure to the pretreated PBS and 6 d of subculture in complete medium, the cells were assayed for plating efficiencies and mutation frequencies (resistance to 6-thioguanine). X-rays (3 Gy) served as a positive control. Cells exposed to insonated PBS with or without ALX or x-rays had statistically significantly elevated mean mutation frequencies (4.37+/-0.97, 4.54+/-1.00, and 24.28+/-3.83 mutant colonies/10(6) viable cells, respectively) relative to corresponding control regimens (ultrasound sham, 2.44+/-0.56; x-ray sham, 2.96+/-0.88 mutant colonies/10(6) viable cells. The data supported the hypothesis that sonochemicals resulting from inertial cavitation have mutagenic potential.

Biophysical effects of high-energy pulsed ultrasound on human cells.

Human benign and malignant cells of different human origin (pancreas, liver, kidney, pharynx, tongue, lip) were exposed to high-energy pulsed ultrasound (HEPUS) in vitro to evaluate the effects of various physical parameters and sonication conditions on cell viability. This included the number of pulses, focal pressure, pulse repetition rate, pulse shape, cell suspension volume, water level of the basin and cell density. Cell viability was found to depend significantly on the number of pulses (exponential), the focal pressure (linear) and the pulse repetition rate (minimum at 1 Hz). Other parameters showed no marked influence. Furthermore, electron microscopy revealed intracellular damage, and proliferation rates of cells surviving sonication were normal after HEPUS exposure. The experimental piezoelectric ultrasound transducer used in the experiments generated oscillating bipolar pulses with high negative pressure amplitudes. Measurements were made of the pulse shape and ultrasonic field of the experimental device and of a conventional lithotripter for comparison.

Nuclear proliferation in syncytia during avian reovirus replication.

Cultured chick embryonic fibroblasts formed syncytia after infection with avian reovirus (ARV) strain 58-132. Mitotic figures were occasionally observed within the syncytia. In addition, many nuclei in the syncytia incorporated 5-bromo-2'-deoxyuridine (BrdU), a DNA replication marker, indicating that they were in the S-phase of the cell cycle. These observations suggested that the nuclei within ARV-induced syncytia originated from nuclear endomitosis without cell division, as well as from cell fusion.