Transgenerational effects of feeding genetically modified maize to nulliparous sows and offspring on offspring growth and health.

This study assessed the effect of feeding genetically modified maize expressing a truncated form of the Cry1Ab protein from Bacillus thuringiensis (Bt MON810 maize) to sows during gestation and lactation and their offspring from weaning to 115 d postweaning on offspring growth and health. After weaning at approximately 28 d of age (d 0), individually penned, mixed sex pigs (approximately 8 kg BW) from sows fed isogenic or Bt maize diets were blocked by sow treatment, sex, and BW and randomly assigned to Bt or isogenic maize diets as follows: i) isogenic maize-fed sow/isogenic maize-fed offspring (iso/iso); ii) isogenic maize-fed sow/Bt maize-fed offspring (iso/Bt); iii) Bt maize-fed sow/isogenic maize-fed offspring (Bt/iso); and iv) Bt maize-fed sow/Bt maize-fed offspring (Bt/Bt). Growth performance was recorded at intervals to harvest at approximately 105 kg BW (n=15/treatment) and blood samples were taken for biochemical analysis on d 0, 30, 70, 100, and 115 postweaning (n=10/treatment). Pigs were harvested on d 115 postweaning (n=10/treatment), and carcass weight, backfat depth, and organ weights (heart, kidney, spleen, and liver) were recorded. Kidney, liver, lymph nodes, and small intestine were collected for histological analysis. Offspring from Bt maize-fed sows were heavier than offspring from isogenic maize-fed sows on d 30 (P<0.05), 100 (P<0.05), and 115 postweaning (P<0.05) and had greater overall ADG (P<0.05). Overall ADFI was greater for offspring from sows fed Bt maize (P<0.05) and for Bt maize-fed pigs (P<0.05). Offspring from Bt maize-fed sows had greater carcass (P<0.05) and lighter spleen (P<0.05) weights. Dressing percentage was greater for Bt maize-fed pigs than isogenic maize-fed pigs (P<0.05), and livers were lighter for pigs in the Bt/Bt group than pigs in the iso/Bt or Bt/iso group (P<0.05). Offspring from Bt maize-fed sows also had greater duodenal crypt depths (P<0.05) and lower villus height/crypt depth ratios (P<0.05). No pathology was observed in the organs, and serum biochemistry values generally remained within normal limits and no overall differences were observed, with the exception of overall γ glutamyltransferase, which was less for pigs on the Bt/Bt treatment than pigs on the iso/Bt and Bt/iso treatments. These results indicate that transgenerational consumption of Bt maize diets is not detrimental to pig growth and health.

Analysis of early changes in the articular cartilage transcriptisome in the rat meniscal tear model of osteoarthritis: pathway comparisons with the rat anterior cruciate transection model and with human osteoarthritic cartilage.

OBJECTIVE: The purpose of this study was to use microarray technology to: (1) understand the early molecular events underlying the damage of articular cartilage initiated by this surgical procedure, and (2) determine whether these changes mimic those that are occurring in human osteoarthritic (OA) cartilage. DESIGN: Cartilage was harvested from both medial and lateral sides of the tibial plateaus and femoral condyles of both meniscal tear (MT) and sham surgery groups on days 3, 7 and 21 post-surgery. mRNA prepared from these rat cartilage samples was used for microarray analysis. RESULTS: Statistical analysis identified 475 genes that were differentially expressed between the sham and MT groups, at one or more of the time points that were analyzed. By integrating these genes with OA-related genes reported previously in a rat OA model and in human OA array studies, we identified 20 commonly changed genes. Six out of these 20 genes (Col5A1, Col6A2, INHBA, LTBP2, NBL1 and SERPINA1) were differentially expressed in two animal models and in human OA. Pathway analysis identified some key features of OA pathology, namely cartilage extracellular matrix remodeling, angiogenesis, and chondrocyte cell death that were recapitulated in the animal models. The rat models suggested increased inflammation and cholesterol metabolic pathways may play important role in early cartilage degeneration. CONCLUSION: We identified a large number of differentially expressed genes in the articular cartilage of the MT model. While there was lack of overall identity in cartilage gene expression between the rat models and human OA, several key biological processes were recapitulated in the rat MT OA model.

Ultrasound monitoring of a novel microwave ablation (MWA) device in porcine liver: lessons learned and phenomena observed on ablative effects near major intrahepatic vessels.

BACKGROUND: Microwave ablation (MWA) is postulated to have several advantages over other thermoablative modalities in the treatment of hepatic tumors. Herein, we use an in vivo porcine model to determine the effect of hepatic blood flow on a novel MWA applicator. METHODS: Four 100-kg pigs underwent hepatic MWA (2,450 MHz, 100 W, 4 min) using a 5.7-mm diameter applicator (Microsulis Americas, Sulis V) inserted near large intrahepatic blood vessels. Real-time monitoring was performed using 3, 5, and 12 MHz diagnostic ultrasound transducers. The ablated zones were sectioned for gross and histological processing. RESULTS: Ablation zones were uniform in shape and size (3-4 cm) and related to power deliver only. Gross and microscopic examination revealed direct extension of ablation zones to the margin of major hepatic blood vessels and occasionally beyond the intended target. Of note, a momentary acoustic white-out occurred around the probe at 25 +/- -1 s in every ablation. DISCUSSION: The Sulis V MWA applicator produced uniform zones of ablation that remain unaffected by convective heat loss. The applicator induced a reproducible but temporary event as seen by ultrasound. Further study is warranted to define the physics, benefits, limits, and clinical safety of this new MWA technology.

Certified reference material for radionuclides in fish flesh sample IAEA-414 (mixed fish from the Irish Sea and North Sea).

A certified reference material (CRM) for radionuclides in fish sample IAEA-414 (mixed fish from the Irish Sea and North Seas) is described and the results of the certification process are presented. Nine radionuclides (40K, 137Cs, 232Th, 234U, 235U, 238U, 238Pu, 239+240Pu and 241Am) were certified for this material. Information on massic activities with 95% confidence intervals is given for six other radionuclides (90Sr, 210Pb(210Po), 226Ra, 239Pu, 240Pu 241Pu). Less frequently reported radionuclides (99Tc, 129I, 228Th, 230Th and 237Np) and information on some activity and mass ratios are also included. The CRM can be used for quality assurance/quality control of the analysis of radionuclides in fish sample, for the development and validation of analytical methods and for training purposes. The material is available from IAEA, Vienna, in 100 g units.

Data-driven analysis approach for biomarker discovery using molecular-profiling technologies.

High-throughput molecular-profiling technologies provide rapid, efficient and systematic approaches to search for biomarkers. Supervised learning algorithms are naturally suited to analyse a large amount of data generated using these technologies in biomarker discovery efforts. The study demonstrates with two examples a data-driven analysis approach to analysis of large complicated datasets collected in high-throughput technologies in the context of biomarker discovery. The approach consists of two analytic steps: an initial unsupervised analysis to obtain accurate knowledge about sample clustering, followed by a second supervised analysis to identify a small set of putative biomarkers for further experimental characterization. By comparing the most widely applied clustering algorithms using a leukaemia DNA microarray dataset, it was established that principal component analysis-assisted projections of samples from a high-dimensional molecular feature space into a few low dimensional subspaces provides a more effective and accurate way to explore visually and identify data structures that confirm intended experimental effects based on expected group membership. A supervised analysis method, shrunken centroid algorithm, was chosen to take knowledge of sample clustering gained or confirmed by the first step of the analysis to identify a small set of molecules as candidate biomarkers for further experimentation. The approach was applied to two molecular-profiling studies. In the first study, PCA-assisted analysis of DNA microarray data revealed that discrete data structures exist in rat liver gene expression and correlated with blood clinical chemistry and liver pathological damage in response to a chemical toxicant diethylhexylphthalate, a peroxisome-proliferator-activator receptor agonist. Sixteen genes were then identified by shrunken centroid algorithm as the best candidate biomarkers for liver damage. Functional annotations of these genes revealed roles in acute phase response, lipid and fatty acid metabolism and they are functionally relevant to the observed toxicities. In the second study, 26 urine ions identified from a GC/MS spectrum, two of which were glucose fragment ions included as positive controls, showed robust changes with the development of diabetes in Zucker diabetic fatty rats. Further experiments are needed to define their chemical identities and establish functional relevancy to disease development.

Determination of self-absorption corrections for gamma analysis of environmental samples: comparing gamma-absorption curves and spiked matrix-matched samples.

Accurate determination of the massic activity of gamma-emitting radionuclides in environmental samples, particularly sediments and soils, cannot be achieved without taking into account sample self-absorption. The extent of self-absorption in the sample will depend on a number of factors including sample composition, density, sample size and gamma-ray energy. The preferred method for correcting for this effect is to use spiked or natural matrix reference materials that match each sample type to be analysed. However, for laboratories that must measure a wide variety of sample matrices this method is too costly and time-consuming. Another technique commonly used is to make direct gamma-ray transmission measurements for each sample. This method, while more practical, still requires a minimum of three measurements to be made for each sample analysed. A more convenient method is to prepare sets of gamma-absorption curves. This approach involves making a series of direct transmission measurements for samples of varying densities but similar type. Sets of matching samples, both spiked and unspiked, were prepared and density correction factors determined using the direct transmission method and the spiked sample approach. It was found that, for soil and sediment samples, these two methods typically differed by 5-9% for higher energy gamma rays and by 12-15% for the 59.54 keV 241Am peak. Gamma-absorption curves were also derived and, for the admittedly limited dataset, 95% confidence intervals of +/-7% for the curve generated using the spiked samples method were obtained.

Devices to improve coronary artery bypass grafting (CABG) surgery.

As off-pump coronary artery bypass grafting becomes the method of choice for cardio-thoracic surgeons, it has become apparent that the facilitation of coronary artery anastomosis on a beating heart needs to be addressed by improved instrumentation. We propose an intraluminal anastomosis device used in conjunction with a biologic-glue to eliminate suturing and serve as a scaffold for constructing stable anastomoses. The device will continue to serve as an eluting stent postoperatively. The simple technique of using the device and the adhesive will require 5 minutes or less for anastomosis. Moreover, we introduce a novel parallel port vacuum stabilizer foot equipped with a uniform lateral tension inducing turnbuckle mechanism to be used with off-pump stabilization systems. A proposed in vitro protocol details using saphenous vein segments and coronary arteries to test the patency of multiple end-to-side grafts. A pressure transducer will be attached to the graft to monitor flow characteristics. An in vivo protocol details construction of anastomoses during off-pump coronary artery bypass grafting in pigs using the anastomosis device, the turnbuckle vacuum foot, and the biologic-glue. Anastomosis patency will be evaluated intraoperatively and one month postoperatively. Furthermore, the graft site will be examined via flow measurement, angiography, and histological analysis.

Gene expression analysis of the acute phase response using a canine microarray.

The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.

Transuranic biokinetic parameters for marine invertebrates--a review.

A catalogue of biokinetic parameters for the transuranic elements plutonium, americium, curium, neptunium, and californium in marine invertebrates is presented. The parameters considered are: the seawater-animal concentration factor (CF); the sediment-animal concentration ratio (CR); transuranic assimilation efficiency; transuranic tissue distribution and transuranic elimination rates. With respect to the seawater-animal CF, authors differ considerably on how they define this parameter and a seven-point reporting system is suggested. Transuranic uptake from sediment by animals is characterised by low CRs. The assimilation efficiencies of transuranic elements in marine invertebrates are high compared to vertebrates and mammals in general and the distribution of transuranics within the body tissue of an animal is dependent on the uptake path. The elimination of transuranics from most species examined conformed to a standard biphasic exponential model though some examples with three elimination phases were identified.

Temporal gene expression analysis of monolayer cultured rat hepatocytes.

The use of cultured primary hepatocytes within toxicology has proven to be a valuable tool for researchers, however, questions remain with regard to functional differences observed in these hepatocytes relative to the intact liver. Cultured hepatocytes have typically been described as dedifferentiated, a classification based upon the investigation of a few key cellular processes or hepatocellular markers. In the present study, parallel expression monitoring of approximately 8700 rat genes was used to characterize mRNA changes over time in hepatocyte cultures using Affymetrix microarrays. We isolated and labeled mRNA from whole rat livers, hepatocyte-enriched cell pellets, and primary cultured hepatocytes (4, 12, 24, 48, and 72 h postplating), and hybridized these samples to microarrays. From these data, several pairwise and temporal gene expression comparisons were made. Gene expression changes were confirmed by RT/PCR and by performing replicate experiments and repeated hybridizations using a rat toxicology sub-array that contained a 900-gene subset of the 8700-gene rat genomic microarray. PCR data qualitatively reproduced the temporal patterns of gene expression observed with microarrays. Cluster analysis of time course data using self-organizing maps (SOM) revealed a classic hepatocyte dedifferentiation response. Functional grouping of genes with similar transcriptional patterns showed time-dependent regulation of phase I and phase II metabolizing enzymes. In general, cytochrome P450 mRNA expression was repressed, but expression of phase II metabolizing enzymes varied by class (upregulation of glucuronidation, downregulation of sulfation). Potential metabolic targets for toxic insult, such as glutathione metabolism, gluconeogenesis, and glycolysis, were also affected at the transcriptional level. Progressive induction of several genes associated with the cellular cytoskeleton and extracellular matrix was observed in accord with physical changes in cell shape and connectivity associated with cellular adhesion. Finally, many transcriptional changes of genes involved in critical checkpoints throughout the hepatocyte cell cycle and differentiation process were observed. In total, these data establish a more comprehensive understanding of hepatocellular dedifferentiation and reveal many novel aspects of physiological and morphological hepatocyte adaptation. An assembly of all transcripts that demonstrated differential expression in this study can be found in the Supporting Information.

Technetium-99 in the Irish marine environment.

Technetium-99 activity concentrations in seawater and biota from Irish coastal waters are presented. Time series measurements of 99Tc in seawater and Fucus vesiculosus from the western Irish Sea show that activity concentrations have increased in line with the increase in discharges of 99Tc from Sellafield. The peak in activity concentrations in both seawater and Fucus vesiculosus occurred in 1997 approximately two years after the peak in 99Tc discharges. The highest activity concentration recorded in Fucus vesiculosus showed a 29-fold increase over the mean concentration for the period 1988-1993. Technetium-99 activity concentrations were measured in fish, lobsters, prawns, mussels and oysters landed at major fishing ports on the east and northeast coasts of Ireland between 1996 and 1998. Concentration factors for 99Tc in the brown seaweed Fucus vesiculosus and certain species of fish, crustaceans and molluscs from the Irish Sea were estimated. In general, these concentration factors were higher than those in the literature which were derived from laboratory studies, but agreed well with values which were based on field studies. The mean committed effective doses to Irish typical and heavy seafood consumers due to 99Tc in the period 1996-1998 were 0.061 and 0.24 microSv, respectively.

Protein oxidation: examination of potential lipid-independent mechanisms for protein carbonyl formation.

Previous data indicated that diquat-mediated protein oxidation (protein carbonyl formation) occurs through multiple pathways, one of which is lipid dependent, and the other, lipid independent. Studies reported here investigated potential mechanisms of the lipid-independent pathway in greater detail, using bovine serum albumin as the target protein. One hypothesized mechanism of protein carbonyl formation involved diquat-dependent production of H2O2, which would then react with site-specifically bound ferrous iron as proposed by Stadtman and colleagues. This hypothesis was supported by the inhibitory effect of catalase on diquat-mediated protein carbonyl formation. However, exogenous H2O2 alone did not induce protein carbonyl formation. Hydroxyl radical-generating reactions may result from the H2O2-catalyzed oxidation of ferrous iron, which normally is bound to protein in the ferric state. Therefore, the possible reduction of site-specifically bound Fe3+ to Fe2+ by the diquat cation radical (which could then react with H2O2) was also investigated. The combination of H2O2 and an iron reductant, ascorbate, however, also failed to induce significant protein carbonyl formation. In a phospholipid-containing system, an ADP:Fe2+ complex induced both lipid peroxidation and protein carbonyl formation; both indices were largely inhibitable by antioxidants. There was no substantial ADP:Fe(2+)-dependent protein carbonyl formation in the absence of phospholipid under otherwise identical conditions. Based on the lipid requirement and antioxidant sensitivity, these data suggest that ADP:Fe(2+)-dependent protein carbonyl formation occurs through reaction of BSA with aldehydic lipid peroxidation products. The precise mechanism of diquat-mediated protein carbonyl formation remains unclear, but it appears not to be a function of H2O2 generation or diquat cation radical-dependent reduction of bound Fe3+.

Transluminal radiofrequency tissue ablation with use of metallic stents.

PURPOSE: To determine if transluminal coagulative necrosis can be induced by applying radiofrequency (RF) energy to indwelling metallic stents. MATERIALS AND METHODS: RF energy was applied to metallic alloy stents (20-68 mm length, 5-16 mm diameter) in tissue phantom (n = 31), ex vivo bovine liver (n = 10), and in vivo porcine hepatic veins (n = 4). For ex vivo and in vivo liver experiments, RF was applied for 5-6 minutes, titrating generator output to produce 85 degrees-95 degrees C temperatures at the stent surface. Local and remote temperature sensing was performed. Imaging and pathologic studies documented the extent of coagulation necrosis. RESULTS: Phantom studies demonstrated uniform temperature distribution along the entire stent length for all Elgiloy stents powered for 2 minutes with a minimum of 120 watts. Shorter stents required less power or reduced time to achieve uniform temperature. In ex vivo liver, 25-mm stents (n = 5) showed 8-10 mm of uniform circumferential coagulation necrosis along the entire stent length. Fifty-millimeter stents showed less uniform coagulation necrosis. For the in vivo stents (20 mm), 8-10 mm of uniform circumferential coagulation necrosis surrounded the stent along its entire length. CONCLUSION: Metallic stents can be used to deliver transluminal RF energy from an external source, inducing heat deposition with resultant circumferential tissue coagulation. Clinical applications might include reduction of intimal proliferation in vascular diseases and/or treatment of periluminal tumors compressing the bile ducts, the urethra, or other luminal structures.

Quantitative assessment of variables that influence soft-tissue electrovaporization in a fluid environment.

OBJECTIVES: To evaluate the process of soft-tissue electrovaporization and to study variables that affect tissue clearance rates in a laboratory setting, in order to identify parameters that can optimize transurethral electrovaporization of the prostate. METHODS: Fresh bovine skeletal muscle, equivalent in impedance and surface properties to the human prostate, was submerged in 3.3% sorbitol solution and electrovaporized with a grooved monopolar electrode attached to the weighted arm of a linear actuator. The effects of excursion rate, applied mechanical load, power setting, electrode configuration, and generator performance on the volume of tissue removed, were assessed. RESULTS: Tissue removal increased significantly when electrode excursion rate was slowed from 25 to 15 mm/s (P < 0.05) and then to 10 mm/s (P < 0.05); when the load was increased from 20 to 50 g (P < 0.005); and when dial power was increased from 120 to 150 W (P < 0.01). Tissue removal was generator dependent. There was no significant difference between the Force 40 and the Force 2 (P > 0.4), but a new computer-controlled constant power output generator (Force FX) did significantly improve tissue vaporization at an equivalent power setting (P < 0.005 and P < 0.01, respectively). Tissue removal was also dependent upon electrode configuration, with the VaporTrode-Grooved Bar removing significantly more tissue than either an ungrooved roller bar of equivalent size or 2-mm smooth roller ball, respectively, both after a single pass (P < 0.001 and P < 0.05) and after five repeated passes (P < 0.05 and P < 0.005). The histologic depth of tissue thermal effect was less than 1 mm, but it was 38% greater for the VaporTrode-Grooved Bar (0.68 mm) than for the standard cutting loop (0.5 mm, P < 0.01). CONCLUSIONS: Using a novel method to quantify tissue removal, we have demonstrated that electrode configuration, excursion rate, applied load, power setting, and generator performance are interdependent factors that influence the efficacy of the electrovaporization process in a fluid environment.

Prostate heating patterns comparing electrosurgical transurethral resection and vaporization: a prospective randomized study.

PURPOSE: A prospective study was performed to determine if transurethral electrosurgical vaporization of the prostate is associated with unseen heat damage to vital periprostatic structures compared to conventional loop resection. In addition, energy consumption and its relationship to observed tissue temperature at the prostate periphery were evaluated for each treatment. MATERIALS AND METHODS: Patients with moderate to severe symptoms of benign prostatic bladder outflow obstruction and objective evidence of diminished flow or acute urinary retention were randomized to undergo either transurethral loop resection or electrosurgical vaporization after stratification for gland volume. Instrumentation was standardized for both groups except for the monopolar electrode used. The radiofrequency power source in the study was a new computer controlled generator with a constant power delivery feature. Regional tissue heating patterns were evaluated with optical fiber probes in real time. Four stationary sites were chosen for temperature measurements, namely the lateral lobe of the prostate, neurovascular bundle beside the prostatic apex at the level of the external sphincter, and anterior rectal wall at the level of the prostatic base and apex. A pull back technique was used to search for hot points in the long axis of the probe (3-dimensional temperature mapping) in 2 patients from each group. Incident generator panel power settings for the electrosurgical vaporization treatments were equivalent to those commonly used for loop resection (150 watts) and were adjusted up or down as needed. RESULTS: Prostate electrosurgical vaporization was possible at generator panel settings that were nearly equivalent to those for transurethral resection of the prostate (130 to 190 watts). No significant rectal or sphincteric heating was detected with either procedure. Conductive cooling of the neurovascular bundles was observed in 2 patients in each group toward the end of the operation. More energy was used per minute of treatment during electrosurgical vaporization than with regular loop resection (p < 0.004) but this was not associated with unwanted tissue heating. CONCLUSIONS: Neither conventional loop resection nor electrosurgical vaporization of the prostate appeared to be unsafe treatments with respect to unseen deep heating effects to vital periprostatic structures when performed at equivalent low incident power. The extra energy used during electrosurgical vaporization provided the benefit of improved coagulative hemostasis concurrently with shallow tissue vaporization using pure cutting current alone, without compromising treatment safety.

Pulmonary ferritin: differential effects of hyperoxic lung injury on subunit mRNA levels.

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Initial in vivo experience with EIT as a thermal estimator during hyperthermia.

Thermal imaging experiments using electrical impedance tomography (EIT) have been conducted during hyperthermia treatments delivered to two human patients and one animal subject. Coplanar and circumferential arrays of 16 and 32 tin-plated copper electrodes etched on a 0.005" polyimide sheet were used to inject 12.5 KHz current patterns of increasing sinusoidal spatial frequencies and subsequent potential distributions were recorded at each electrode site. Image reconstruction was achieved with a finite element method and difference images of conductivity changes during the course of treatment were formed. An assumed linear relationship (2%/degree C increase) between tissue impedance change and temperature change was used to produce thermal images of the treatment field in patients whereas an empirically measured nonlinear relationship obtained from excised tissue samples was applied retrospectively in the animal subject case. Reconstructed conductivity changes are shown to be possible given electrical data measured in vivo during hyperthermia delivery with conventional equipment (spiral microstrip applicator at 433 MHz). These correlated well with direct temperature measurements and demonstrated quantitative levels of agreement to the extent that estimated temperature accuracies were approximately 1.5 degrees C; although large errors (> 5 degrees C) did exist. This work suggests that EIT is a potentially useful tool for hyperthermia treatment monitoring and assessment. The relationship between tissue impedance and temperature is complex and confounds the ability to make simple correlations between conductivity and temperature changes. Further, study is required to discern whether this will ultimately limit EIT as a thermal estimator or whether it will lead to more fundamental uses of impedance as an indicator of thermal effect.

Factors influencing electrovaporization in the treatment of benign prostatic hyperplasia.

Electrovaporization refers to the process of vaporizing tissue using electrical energy. Proposed as a new treatment method for benign prostatic hyperplasia (BPH), this technique allows removal of prostatic tissue with simultaneous coagulation, thereby minimizing blood loss. The recent increase in popularity of electrovaporization in the treatment of BPH warrants a quantitative assessment of the process, including an objective evaluation of its influencing factors. In this study, the effects of power, mechanical loading, and excursion rate on tissue removal by electrovaporization were examined in fresh skeletal muscle. A monopolar electrode was attached to the weighted arm of a linear motion system and rolled across the tissue surface while activated by a radio-frequency generator. The tissue samples were frozen and cut longitudinally to allow visualization and measurement of the vaporized groove using an optical imaging technique. The volume of tissue removed increased significantly when power was increased from 120 to 150 W (46 to 119 mm3, p = 0.006), when the load was increased from 20 to 50 gm (20 to 119 mm3, p = 0.002), and when the excursion rate was decreased from 25 to 15 mm/s (29 to 69 mm3, p < 0.05) and from 15 to 10 mm/s (69 to 137 mm3, p < 0.05). There was no significant gain in volume removed when power was increased to 180 W or when the load was increased to 70 gm, indicating that these factors are constrained with regard to optimal tissue removal. Using a novel method to quantitatively assess tissue removal by electrovaporization, this study has demonstrated that greater tissue removal can be achieved by increasing power, increasing the load, or decreasing the excursion rate, but only within limits.

Tissue impedance as a function of temperature and time.

Tissue impedance dependence on temperature has been measured for six tissue types. The information was gathered using an automated laboratory under computer control. This information is needed to be able to input these values into a computer model that predicts temperature distribution produced by delivery of radio frequency energy. Due to the thermal dose of time and temperature, tissue properties change and no published data are available that document this. Since there are no theoretical predictions, empirical data were measured to supply this information. Using an aluminum cylindrical cavity of volume 1.69 cm3, muscle, liver, brain, and fat tissue impedance were measured at 500 kHz over a range of temperatures. All tests began at room temperature where baseline measurements were made. The cylinder was then placed in a constant temperature water bath at between 30 and 90 degrees C. The tests were run for a period of 10 to 30 minutes. Temperature homogeneity was carefully studied throughout the volume of the cylinder. It was found that thermal equilibrium occurred within four minutes. Special care was taken with the tissue sample in regard to optimize moisture and freshness, and minimize fat content. Grain orientation was also taken into consideration depending on the test. For all tissue types, resistivity decreased initially as the sample temperature equilibrated with the bath temperature. For temperatures less than 75 degrees C, resistivity values remained approximately constant over time.

Diquat-dependent protein carbonyl formation. Identification of lipid-dependent and lipid-independent pathways.

In a previous report on diquat-dependent oxidative damage in rat hepatic microsomes, protein oxidation, as measured by protein carbonyl (PC) formation, was observed in addition to lipid peroxidation (LP). Both phenomena were antioxidant sensitive. Inhibition of PC formation was somewhat surprising given the proposed mechanism of metal-catalyzed protein oxidation. Studies reported here examined diquat-dependent PC formation in greater detail. In rat hepatic microsomes, diquat-dependent thiobarbituric acid-reactive substances (TBARS) and PC formation were time and concentration dependent. In this system, LP was inhibited completely by U-74006F or U-78517G, whereas PC formation was inhibited only partially by these antioxidants. In an essentially lipid-free system consisting of purified rat hepatic cytochrome P450 reductase, BSA and an NADPH-generating system, PC formation was also observed, but was not antioxidant-sensitive. Under these conditions, minimal diquat-dependent TBARS formation was observed. The observation of relative antioxidant insensitivity is consistent with H2O2 (generated during the diquat redox cycle) catalyzing protein oxidation via a site-specific, metal-catalyzed mechanism. Thus, different pathways would appear to be involved in diquat-dependent PC formation in lipid-containing and lipid-free systems. Carbon tetrachloride induces LP following reductive activation to the trichloromethyl free radical, a pathway not directly involving H2O2 generation. In the microsomal system, CCl4 induced TBARS and PC formation, both of which were completely inhibitable by antioxidants. Taken together, these data suggest that diquat induces PC formation by lipid-dependent (antioxidant-sensitive) and lipid-independent (antioxidant-insensitive) pathways. In microsomes, both pathways contribute to diquat-dependent PC formation. Data for the lipid-independent pathway are consistent with the mechanism of metal-catalyzed protein oxidation proposed by Stadtman and colleagues (reviewed in Free Radic Biol Med 9: 315-325, 1990), while the lipid-dependent pathway is likely secondary to LP itself--via a Michael-type addition reaction between hydroxyalkenals and protein sulfhydryl groups, amino groups or other protein nucleophiles. The latter pathway is also responsible for carbon tetrachloride-dependent PC formation. Additional studies are in progress to further characterize the lipid-independent mechanism.