Timothy D. Whitehead

Gene expression does not change significantly in C3h 10T½ cells after exposure to 847.74 CDMA or 835.62 FDMA radiofrequency radiation

Abstract Whitehead, T. D., Moros, E. G., Brownstein, B. H. and Roti Roti, J. L. Gene Expression does not Change Significantly in C3H 10T ½ Cells after Exposure to 847.74 CDMA or 835.62 FDMA Radiofrequency Radiation. Radiat. Res. 165, 626–635 (2006). In vitro experiments with C3H 10T½ mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip® to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip® data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.

Expression of the Proto-oncogene Fos after Exposure to Radiofrequency Radiation Relevant to Wireless Communications

Abstract Whitehead, T. D., Brownstein, B. H., Parry, J. J., Thompson, D., Cha, B. A., Moros, E. G., Rogers, B. E. and Roti Roti, J. L. Expression of the Proto-oncogene Fos after Exposure to Radiofrequency Radiation Relevant to Wireless Communications. Radiat. Res. 164, 420–430 (2005). In this study the expression levels of the proto-oncogene Fos were measured after exposure to radiofrequency (RF) radiation at two relatively high specific absorption rates (SARs) of 5 and 10 W/kg for three types of modulated signals: 847.74 MHz code division multiple access (CDMA), 835.62 MHz frequency division multiple access (FDMA), and 836.55 MHz time division multiple access (TDMA). This work was undertaken to confirm a previous report by Goswami et al. (Radiat. Res. 151, 300–309, 1999) that CDMA and FDMA radiation caused small but statistically significant increases in Fos levels as cells entered plateau phase during exposure. No effects on Myc or Jun levels were observed in that study. Therefore, in the present study, analyses were restricted to Fos expression during the transition from exponential growth to plateau phase. Fos expression was measured using the real-time polymerase chain reaction (RT-PCR) technique. Serum-stimulated C3H 10T½ cells were used as a positive control for Fos expression. Possible influences of final cell number or pH variability on Fos expression were evaluated. Expression of Fos mRNA in C3H 10T½ cells was not significantly different from that found after sham exposure at either SAR level for any signal modulation. Therefore, the results of Goswami et al. could not be confirmed.

The effects of 41°C hyperthermia on the DNA repair protein, MRE11, correlate with radiosensitization in four human tumor cell lines

Purpose: The goal of this study was to determine if reduced availability of the DNA repair protein, MRE11, for the repair of damaged DNA is a basis for thermal radiosensitization induced by moderate hyperthermia. To test this hypothesis, we measured the total amount of MRE11 DNA repair protein and its heat-induced alterations in four human tumor cell lines requiring different heating times at 41°C to induce measurable radiosensitization. Materials and methods: Human colon adenocarcinoma cell lines (NSY42129, HT29 and HCT15) and HeLa cells were used as the test system. Cells were irradiated immediately after completion of hyperthermia. MRE11 levels in whole cell extract, nuclear extract and cytoplasmic extracts were measured by Western blotting. The nuclear and cytoplasmic extracts were separated by TX100 solubility. The subcellular localization of MRE11 was determined by immunofluorescence staining. Results: The results show that for the human tumor cell lines studied, the larger the endogenous amount of MRE11 protein per cell, the longer the heating time at 41°C required for inducing measurable radiosensitization in that cell line. Further, the residual nuclear MRE11 protein level, measured in the nuclear extract and in the cytoplasmic extract as a function of heating time, both correlated with the thermal enhancement ratio (TER). Conclusions: These observations are consistent with the possibility that delocalization of MRE11 from the nucleus is a critical step in the radiosensitization by moderate hyperthermia.

An animal model with a cardiomyocyte-specific deletion of estrogen receptor alpha: functional, metabolic, and differential network analysis.

Estrogen exerts diverse biological effects in multiple tissues in both animals and humans. Much of the accumulated knowledge on the role of estrogen receptor (ER) in the heart has been obtained from studies using ovariectomized mice, whole body ER gene knock-out animal models, ex vivo heart studies, or from isolated cardiac myocytes. In light of the wide systemic influence of ER signaling in regulating a host of biological functions in multiple tissues, it is difficult to infer the direct role of ER on the heart. Therefore, we developed a mouse model with a cardiomyocyte-specific deletion of the ERα allele (cs-ERα−/−). Male and female cs-ERα−/− mice with age/sex-matched wild type controls were examined for differences in cardiac structure and function by echocardiogram and differential gene expression microarray analysis. Our study revealed sex-differences in structural parameters in the hearts of cs-ERα−/− mice, with minimal functional differences. Analysis of microarray data revealed differential variations in the expression of 208 genes affecting multiple transcriptional networks. Furthermore, we report sex-specific differences in the expression of 56 genes. Overall, we developed a mouse model with cardiac-specific deletion of ERα to characterize the role of ERα in the heart independent of systemic effects. Our results suggest that ERα is involved in controlling the expression of diverse genes and networks in the cardiomyocyte in a sex-dependent manner.

Sexual dimorphism in myocardial acylcarnitine and triglyceride metabolism

BackgroundCardiovascular disease is the leading cause of death among diabetic patients. Importantly, recent data highlight the apparent sexual dimorphism in the pathogenesis of cardiovascular disease in diabetics with respect to both frequency- and age-related risk factors. The disposition to cardiovascular disease among diabetic patients has been attributed, at least in part, to excess lipid supply to the heart culminating in lipotoxicity of the heart and downstream derangements. A confounding factor in obese animal models of diabetes is that increased peripheral lipid availability to the heart can induce cardio-metabolic remodeling independent of the underlying pathophysiology of diabetes, thus masking the diabetic phenotype. To that end, we hypothesized that the use of non-obese diabetic (NOD) animal models will reveal metabolic signatures of diabetes in a sex-specific manner.MethodsTo test this hypothesis, male and female NOD Goto-Kakizaki (GK) rats were used to assess the expression profile of 84 genes involved in lipid metabolism. In parallel, targeted lipidomics analysis was performed to characterize sex differences in homeostasis of non-esterified fatty acids (NEFA), acylcarnitines (AC), and triglycerides (TG).ResultsOur analysis revealed significant sex differences in the expression of a broad range of genes involved in transport, activation, and utilization of lipids. Furthermore, NOD male rats exhibited enhanced oxidative metabolism and accumulation of TG, whereas female NOD rats exhibited reduced TG content coupled with accumulation of AC species. Multi-dimensional statistical analysis identified saturated AC16:0, AC18:0, and AC20:0 as dominant metabolites in mediating sex differences in AC metabolism. Confocal microscopy of rat cardiomyocytes exposed to AC14:0, AC16:0, and AC18:0 confirmed induction of ROS with AC18:0 being more potent followed by AC14:0.ConclusionOverall, we demonstrate sex differences in myocardial AC and TG metabolism with implications for therapy and diagnosis of diabetic cardiovascular disease.

A PET-Compatible Tissue Bioreactor for Research, Discovery, and Validation of Imaging Biomarkers and Radiopharmaceuticals: System Design and Proof-of-Concept Studies

Research and discovery of novel radiopharmaceuticals and targets thereof generally involves initial studies in cell cultures, followed by animal studies, both of which present several inherent limitations. The objective of this work was to develop a tissue bioreactor (TBR) enabling modulation of the microenvironment and to integrate the TBR with a small-animal PET scanner to facilitate imaging biomarker research and discovery and validation of radiopharmaceuticals. Methods: The TBR chamber is a custom-blown, water-jacketed, glass vessel enclosed in a circulating perfusion bath powered by a peristaltic pump, which is integrated within the field of view of the PET scanner. The chamber is in series with a gas exchanger and a vessel for degassing the system during filling. Dissolved oxygen/temperature probes and septa for injection or sampling are located at the inlet and outlet of the cell chamber. A pH probe is located at the chamber outlet. Effluent is collected in the fraction collector as mixed-cup samples. In addition, both medium and tissue chamber can be sampled to investigate tissue and secretory products through multiscale analysis. As a proof of concept, we studied the effects of lipids on glucose uptake using HepG2 cells. To that end, we varied the nutrient substrate environment over a period of approximately 27 d, before and after the addition of lipids, and studied the effects of pioglitazone, a peroxisome proliferator-activated receptor γ agonist, on lipid and glucose uptake. In parallel, the TBR was imaged by PET in conjunction with 11C-palmitate in the presence and absence of lipids to characterize 11C-palmitate uptake. Results: The O2 consumption, glucose consumption, lactate production, and free fatty acid consumption and production rates were consistent in demonstrating the effects of lipids on glucose uptake. Pioglitazone exhibited improved glucose uptake within 3 d of treatment. Semiquantitative analysis suggested that lipids induced greater 11C-palmitate uptake. Conclusion: The integrated TBR offers a platform to monitor and modulate the tissue microenvironment, thus facilitating tissue-specific imaging and therapeutic biomarkers of disease, identification of molecular diagnostic markers, and validation of radiopharmaceuticals in both rodent and human cell lines.

Artificial tissue bioreactor (ATB) for biological and imaging applications

Three-dimensional (3D) culture systems are increasingly applied to study tissue biology. In this work, we report on the development of an artificial tissue bioreactor (ATB) designed to simulate the 3D structure and microenvironment of tissues in vivo, with multiple avenues of sampling, including the tissue chamber, for downstream analysis. Additionally, the ATB is integrated with the microPET Focus F220 for in-vivo imaging applications. As a proof-of-concept, we characterized the effects of lipids on glucose utilization using HepG2 cells. ATB studies were performed pre- and post- therapeutic intervention with the PPAR-γ agonist pioglitazone. In parallel, Glucose Tolerance Test (GTT) is performed on media samples to assess glucose uptake by cells as a measures of insulin signaling sensitivity. Fatty acid uptake in the ATB cell chamber is measured using [11C]Palmitate with microPET imaging. Overall, the ATB will facilitate the use of existing and novel radiopharmaceuticals in discovery of validating and translating insights derived from ATB studies to pre-clinical animal studies, to clinical evaluation.