Noninvasive magnetic resonance imaging of the development of individual colon cancer tumors in rat liver

Monitoring tumor development is essential for the understanding of mechanisms involved in tumor progression and to determine efficacy of therapy. One of the evolving approaches is longitudinal noninvasive magnetic resonance imaging (MRI) of tumors in experimental models. We applied high-resolution MRI at 7 Tesla to study the development of colon cancer tumors in rat liver. MRI acquisition was triggered to the respiratory cycle to minimize motion artifacts. A special radio frequency (RF) coil was designed to acquire detailed T1-weighted and T2-weighted images of the liver. T2-weighted images identified hyperintense lesions representing tumors with a minimum diameter of 2 mm, enabling the determination of growth rates and morphological aspects of individual tumors. It is concluded that high-resolution MRI using a dedicated RF coil and triggering to the respiratory cycle is an excellent tool for quantitative and morphological analysis of individual diffusely distributed tumors throughout the liver. However, at present, MRI requires expensive equipment and expertise and is a time-consuming methodology. Therefore, it should preferably be used for dedicated applications rather than for high-throughput assessment of total tumor load in animals.     

基于超声射频信号的大鼠脂肪肝分级诊断研究

目的：从分析肝脏超声射频信号的角度,探究一种对脂肪肝进行分级定量诊断的新方法。方法：80只Wistar大鼠,通过喂养高脂饲料建立大鼠脂肪肝模型,其中正常肝的大鼠28只,轻度脂肪肝的大鼠21只,中度脂肪肝的18只,重度脂肪肝的13只。然后采集大鼠左右肝的超声射频信号,选取大鼠肝脏部位感兴趣区域的超声射频信号,分析大鼠肝脏超声射频信号的脂肪肝特征信息,提取了射频信号幅度包络值的均值／标准差（MSR）、偏度（SK）、峰度（Ku）这三个统计特征量,再利用BP神经网络对大鼠脂肪肝进行分类识别。结果：该方法对大鼠正常肝的识别率达92．5％,轻度脂肪肝的识别率达87．5％,中度脂肪肝的识别率达76．7％．重度脂肪肝的识别率达77．3％。结论：本文研究的方法可对大鼠脂肪肝进行分级定量诊断,且证明了超声射频信号在脂肪肝诊断中是有价值的,为对分级诊断脂肪肝疾病的研究提供了新的方向。     

Time-restricted feeding entrains daily rhythms of energy metabolism in mice

Energy metabolism, oxygen consumption rate (VO2), and respiratory quotient (RQ) in mice were monitored continuously throughout 12:12-h light-dark cycles before, during, and after time-restricted feeding (RF). Mice fed ad libitum showed robust daily rhythms in both parameters: high during the dark phase and low during the light phase. The daily profile of energy metabolism in mice under daytime-only feeding was reversed at the beginning of the first fasting night. A few days after daytime-only feeding began, RF also reversed the circadian core body temperature rhythm. Moreover, RF for 6 consecutive days shifted the phases of circadian expression patterns of clock genes in liver significantly by 8-10 h. When mice were fed a high-fat (HF) diet ad libitum, the daily rhythm of RQ dampened day by day and disappeared on the sixth day of RF, whereas VO2 showed a robust daily rhythm. Mice fed HF only in the daytime had reversed VO2 and RQ rhythms. Similarly, mice fed HF only in the daytime significantly phase shifted the clock gene expression in liver, whereas ad libitum feeding with HF had no significant effect on the expression phases of liver clock genes. These results suggested that VO2 is a sensitive indicator of entrainment in the mouse liver. Moreover, physiologically, it can be determined without any surgery or constraint. On the basis of these results, we hypothesize that a change in the daily VO2 rhythm, independent of the energy source, might drive phase shifts of circadian oscillators in peripheral tissues, at least in the liver.     

Relationship between FGF21 and UCP1 levels under time-restricted feeding and high-fat diet

Fibroblast growth factor 21 (FGF21) exhibits a circadian oscillation, and its induction is critical during fasting. When secreted by liver and skeletal muscle, FGF21 enhances thermogenic activity in brown adipose tissue (BAT) by utilizing uncoupling protein 1 (UCP1) to dissipate energy as heat. Recently, it has been reported that UCP1 is not required for FGF21-mediated reduction in body weight or improvements in glucose homeostasis. As the relationship between FGF21 and UCP1 induction in tissues other than BAT is less clear, we tested the effect of restricted feeding (RF) and high dietary fat on FGF21 circadian expression and its correlation with UCP1 expression in liver and white adipose tissue (WAT). High dietary fat disrupted Fgf21 mRNA circadian oscillation but increased its levels in WAT. RF led to increased liver FGF21 protein levels, whereas those of UCP1 decreased. In contrast, WAT FGF21 protein levels increased under high-fat diet, whereas those of UCP1 decreased under RF. In summary, FGF21 exhibits circadian oscillation, which is disrupted with increased dietary fat. The relationship between FGF21 and UCP1 levels depends on the tissue and the cellular energy status.     

Immunocytochemistry of Kaposi's sarcoma-like tumor cells from pigtailed macaques with simian AIDS

Some macaques infected with SRV-2 developed SAIDS and RF, a Kaposi's sarcoma (KS)-like tumor. We investigated the immunophenotypic markers of this SAIDS-associated retroperitoneal fibromatosis (RF). RF tumor is characterized by proliferation of spindle cells accompanying inflammatory cell infiltrates, fibroblasts, and endothelial cells. RF spindle cells in tumor tissues revealed several immunobiologic characteristics similar to vascular smooth-muscle cells or myofibroblasts based on positive immunoreactivity of smooth-muscle α-actin and desmin. The majority of cultured RF spindle cells also expressed specific markers for vascular smooth muscle. These results suggest that the RF spindle cells are derived from vascular smooth-muscle cells. Furthermore, RF tissues and cells were persistently infected with SRV-2, which may play an important role in viral etiology of AIDS-associated neoplasm in this macaque model.     

Riboflavin uptake by the human-derived liver cells Hep G2: Mechanism and regulation

The water-soluble vitamin riboflavin (RF) plays a critical role in many metabolic reactions, and thus, is essential for normal cellular functions and growth. The liver plays a central role in normal RF metabolism and is the site of maximal utilization of the vitamin. The mechanism of liver uptake of RF has been studied in animals, but no information is available describing the mechanism of the vitamin uptake in the human situation and its cellular regulation. In this study, we used the human-derived liver cells Hep G2 as an in vitro model system to address these issues. Uptake of RF by Hep G2 cells was found to be temperature- and energy-dependent but Na⁺-independent in nature. Uptake seemed to involve a carrier-mediated process as indicated by the saturation as a function of substrate concentration (apparent K_m 0.41 ± 0.08 μM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process [inhibition constant (K_i) of 1.84 and 6.32 μM, respectively]. RF uptake was energy dependent, and was inhibited by the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (K_i of 0.10 mM). Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, and protein tyrosine kinase (PTK)-mediated pathways did not affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of Ca²⁺/calmodulin-mediated pathway significantly inhibited the uptake process; this effect seemed to be mediated through a decrease in the V_max of the substrate uptake process. Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant up-regulation in the substrate uptake; this effect was specific for RF and was mediated mainly by means of an increase in the V_max of the uptake process. These results describe, for the first time, the mechanism and cellular regulation of RF uptake by a human-derived liver cellular preparation, and shows the involvement of a carrier-mediated system in the uptake process. Furthermore, the uptake process seems to be regulated by an intracellular Ca²⁺/calmodulin-mediated pathway and by extracellular substrate levels. J. Cell. Physiol. 176:588–594, 1998. © 1998 Wiley-Liss, Inc.     

No effects of radiofrequency radiation on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone-induced tumorigenesis in female Wistar rats

This study evaluated possible effects of radiofrequency (RF) radiation on tumorigenesis induced by the mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) given in drinking water. Female Wistar rats aged 7 weeks at the beginning of the experiments were randomly divided into four groups of 72 animals: a cage-control group and three MX-exposed groups (a daily average dose of 1.7 mg MX/kg body weight for 104 weeks), of which two were exposed to 900 MHz pulsed RF radiation and the third served as a sham-RF-radiation group. The RF-radiation groups were exposed 2 h per day, 5 days per week for 104 weeks at nominal whole-body average SARs of 0.3 W/kg and 0.9 W/kg. Complete histopathology was performed on the rats of the three MX-exposed groups. The tumor types and incidences observed in the MX-exposed animals were similar to those reported earlier in MX-exposed female Wistar rats. RF radiation did not statistically significantly affect mortality or organ-specific incidence of any tumor type. The only statistically significant difference was an increase in the combined frequency of vascular tumors of the mesenteric lymph nodes in the high-RF-radiation group compared to the sham-RF-radiation group. However, additional histopathological analysis of the cage-control animals suggested that this difference was due to unusually low frequency of this type of tumor in the sham-RF-radiation group rather than a high frequency in the high-RF-radiation group. With respect to non-neoplastic findings, statistically significant differences between the RF-radiation groups and the sham-RF-radiation group were observed only for single findings in the lacrimal glands, lungs, liver and skin. Such changes are commonly seen in aged rats and were considered to be unrelated to RF radiation. The results of the present study do not support co-carcinogenic effects of low-level long-term RF-radiation exposure in rats.     

Effects of a brain tumor in a dispersive human head on SAR and temperature rise distributions due to RF sources at 4G and 5G frequencies

In this paper, effects of a brain tumor located in a dispersive human head model on specific absorption rate (SAR) and temperature rise distributions due to different types of RF sources at 4G and 5G cellular frequencies are investigated with the use of a multiphysics model. This multiphysics model analyzes the dispersive human head with the brain tumor and provides the SAR and temperature rise distributions in the head due to the RF source operated at 4G and 5G cellular frequencies in a single finite-difference time-domain simulation. An adjacent antenna operated at 4G and 5G cellular frequencies to the human head is considered as the RF source for near-field exposure, while a plane wave field radiated by base stations operated at 4G and 5G cellular frequencies is considered as the RF source for far-field exposure. Numerical results show that the brain tumor in the head slightly affects the SAR and temperature rise distributions due to different RF sources at 4G and 5G cellular frequencies.     

Caffeine alters circadian rhythms and expression of disease and metabolic markers

The circadian clock regulates many aspects of physiology, energy metabolism, and sleep. Restricted feeding (RF), a regimen that restricts the duration of food availability entrains the circadian clock. Caffeine has been shown to affect both metabolism and sleep. However, its effect on clock gene and clock-controlled gene expression has not been studied. Here, we tested the effect of caffeine on circadian rhythms and the expression of disease and metabolic markers in the serum, liver, and jejunum of mice supplemented with caffeine under ad libitum (AL) feeding or RF for 16 weeks. Caffeine significantly affected circadian oscillation and the daily levels of disease and metabolic markers. Under AL, caffeine reduced the average daily mRNA levels of certain disease and inflammatory markers, such as liver alpha fetoprotein (Afp), C-reactive protein (Crp), jejunum alanine aminotransferase (Alt), growth arrest and DNA damage 45β (Gadd45β), Interleukin 1α (Il-1α), Il-1β mRNA and serum plasminogen activator inhibitor 1 (PAI-1). Under RF, caffeine reduced the average daily levels of Alt, Gadd45β, Il-1α and Il-1β mRNA in the jejunum, but not in the liver. In addition, caffeine supplementation led to decreased expression of catabolic factors under RF. In conclusion, caffeine affects circadian gene expression and metabolism possibly leading to beneficial effects mainly under AL feeding.     

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.     

Chemotherapy and Radiofrequency-Induced Mild Hyperthermia Combined Treatment of Orthotopic Pancreatic Ductal Adenocarcinoma Xenografts

Patients with pancreatic ductal adenocarcinomas (PDAC) have one of the poorest survival rates of all cancers. The main reason for this is related to the unique tumor stroma and poor vascularization of PDAC. As a consequence, chemotherapeutic drugs, such as nab-paclitaxel and gemcitabine, cannot efficiently penetrate into the tumor tissue. Non-invasive radiofrequency (RF) mild hyperthermia treatment was proposed as a synergistic therapy to enhance drug uptake into the tumor by increasing tumor vascular inflow and perfusion, thus, increasing the effect of chemotherapy. RF-induced hyperthermia is a safer and non-invasive technique of tumor heating compared to conventional contact heating procedures. In this study, we investigated the short- and long-term effects (~20 days and 65 days, respectively) of combination chemotherapy and RF hyperthermia in an orthotopic PDAC model in mice. The benefit of nab-paclitaxel and gemcitabine treatment was confirmed in mice; however, the effect of treatment was statistically insignificant in comparison to saline treated mice during long-term observation. The benefit of RF was minimal in the short-term and completely insignificant during long-term observation.     

Effect of input waveform pattern and large blood vessel existence on destruction of liver tumor using radiofrequency ablation: finite element analysis

Much research has been directed at improving the effectiveness of the radiofrequency (RF) ablation of hepatocellular carcinomas. In that point of view, this study was performed to provide comprehensive information of the relation between RF waveforms and thermodynamic response of the tissue with the consideration of four different types of RF waveforms (half-sine, half-square, half-exponential, and damped-sine) to maximize the amount of tumor tissue removed while maintaining the advantages of RF ablation. For the aim of this study, finite element models incorporating results from previous numerical models were used and validated with ex vivo experiments. From analyses of the entire results, we concluded that this study may prove valuable as a first step in providing comprehensive information of the relation between various RF waveforms and thermodynamic responses within the tissue during the RF ablation process. This study may also contribute toward studies to determine an optimum RF waveform capable of maximizing the amount of tumor tissue removed while maintaining the advantages of RF ablation.     

A theoretical study of the effects of RF fields in the vicinity of membranes

In this article the forces associated with the gradients of a radio frequency (RF) field at the boundary between fluids and cell membranes are calculated, and it is shown that they can be large enough to affect the particle motion by amounts that are on the same order of magnitude as the random diffusion motion when the energy imparted to the particles is a reasonable fraction of the thermal energy. The induced dipole moment is assumed to track the alternating RF so that the force exerted by the gradient is in a constant direction; and this in turn leads to a modification of the particle distribution, even when the energy added to the particle is very small. For RF fields of 45 V/m the energy acquired by an induced dipole moment is expected to be on the order of a micro electron volt and small compared to the average thermal energy.     

Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.     

Repeated exposure of C3H/HeJ mice to ultra-wideband electromagnetic pulses: lack of effects on mammary tumors

It has been suggested that chronic, low-level exposure to radiofrequency (RF) radiation may promote the formation of tumors. Previous studies, however, showed that low-level, long-term exposure of mammary tumor-prone mice to 435 MHz or 2450 MHz RF radiation did not affect the incidence of mammary tumors. In this study, we investigated the effects of exposure to a unique type of electromagnetic energy: pulses composed of an ultra-wideband (UWB) of frequencies, including those in the RF range. One hundred C3H/HeJ mice were exposed to UWB pulses (rise time 176 ps, fall time 3.5 ns, pulse width 1.9 ns, peak E-field 40 kV/m, repetition rate 1 kHz). Each animal was exposed for 2 min once a week for 12 weeks. One hundred mice were used as sham controls. There were no significant differences between groups with respect to incidence of palpated mammary tumors, latency to tumor onset, rate of tumor growth, or animal survival. Histopathological evaluations revealed no significant differences between the two groups in numbers of neoplasms in all tissues studied (lymphoreticular tissue, thymus, respiratory, digestive and urinary tracts, reproductive, mammary and endocrine systems, and skin). Our major finding was the lack of effects of UWB-pulse exposure on promotion of mammary tumors in a well-established animal model of mammary cancer.     

Systematic review of wireless phone use and brain cancer and other head tumors

We conducted a systematic review of scientific studies to evaluate whether the use of wireless phones is linked to an increased incidence of the brain cancer glioma or other tumors of the head (meningioma, acoustic neuroma, and parotid gland), originating in the areas of the head that most absorb radiofrequency (RF) energy from wireless phones. Epidemiology and in vivo studies were evaluated according to an agreed protocol; quality criteria were used to evaluate the studies for narrative synthesis but not for meta-analyses or pooling of results. The epidemiology study results were heterogeneous, with sparse data on long-term use (≥ 10 years). Meta-analyses of the epidemiology studies showed no statistically significant increase in risk (defined as P < 0.05) for adult brain cancer or other head tumors from wireless phone use. Analyses of the in vivo oncogenicity, tumor promotion, and genotoxicity studies also showed no statistically significant relationship between exposure to RF fields and genotoxic damage to brain cells, or the incidence of brain cancers or other tumors of the head. Assessment of the review results using the Hill criteria did not support a causal relationship between wireless phone use and the incidence of adult cancers in the areas of the head that most absorb RF energy from the use of wireless phones. There are insufficient data to make any determinations about longer-term use (≥ 10 years).     

Therapeutic Efficacy of Combining PEGylated Liposomal Doxorubicin and Radiofrequency (RF) Ablation: Comparison between Slow-Drug-Releasing,Non-Thermosensitive and Fast-Drug-Releasing,Thermosensitive Nano-Liposomes

Aims

To determine how the accumulation of drug in mice bearing an extra-hepatic tumor and its therapeutic efficacy are affected by the type of PEGylated liposomal doxorubicin used, treatment modality, and rate of drug release from the liposomes, when combined with radiofrequency (RF) ablation.

Materials and Methods

Two nano-drugs, both long-circulating PEGylated doxorubicin liposomes, were formulated: (1) PEGylated doxorubicin in thermosensitive liposomes (PLDTS), having a burst-type fast drug release above the liposomes’ solid ordered to liquid disordered phase transition (at 42°C), and (2) non-thermosensitive PEGylated doxorubicin liposomes (PLDs), having a slow and continuous drug release. Both were administered intravenously at 8 mg/kg doxorubicin dose to tumor-bearing mice. Animals were divided into 6 groups: no treatment, PLD, RF, RF+PLD, PLDTS, and PLDTS+RF, for intra-tumor doxorubicin deposition at 1, 24, and 72 h post-injection (in total 41, mice), and 31 mice were used for randomized survival studies.

Results

Non-thermosensitive PLD combined with RF had the least tumor growth and the best end-point survival, better than PLDTS+RF (p<0.005) or all individual therapies (p<0.001). Although at 1 h post-treatment the greatest amount of intra-tumoral doxorubicin was seen following PLDTS+RF (p<0.05), by 24 and 72 h the greatest doxorubicin amount was seen for PLD+RF (p<0.05); in this group the tumor also has the longest exposure to doxorubicin.

Conclusion

Optimizing therapeutic efficacy of PLD requires a better understanding of the relationship between the effect of RF on tumor microenvironment and liposome drug release profile. If drug release is too fast, the benefit of changing the microenvironment by RF on tumor drug localization and therapeutic efficacy may be much smaller than for PLDs having slow and temperature-independent drug release. Thus the much longer circulation time of doxorubicin from PLD than from PLDTS may be beneficial in many therapeutic instances, especially in extra-hepatic tumors.     

Estimation of the SAR in the human head and body due to radiofrequency radiation exposure from handheld mobile phones with hands-free accessories

It was reported by others that hands-free accessories increase the absorption of RF energy in a human head compared to a handset alone. The results of this study show that the opposite is observed when proper dosimetric methods are employed. It is pointed out that for correct estimation of the exposure level it is necessary to use appropriate physical and experimental models and measurement instrumentation, following internationally recommended standards. The human phantoms used for measurements involving the hands-free accessories should include the torso; i.e., measurements should not be performed on the head phantom alone. This has a significant impact on the results because the RF energy coupled into the leads of hands-free accessories is strongly attenuated by the body. Numerical simulations using the Finite-Difference Time-Domain (FDTD) method and experimental measurements with a miniature electric-field probe are in good agreement and show a decrease, not an increase, in RF energy exposure in the human head from hands-free accessories.     

Carcinogenicity study of GSM and DCS wireless communication signals in B6C3F1 mice

The purpose of this study using a total of 1170 B6C3F1 mice was to detect and evaluate possible carcinogenic effects in mice exposed to radio-frequency-radiation (RFR) from Global System for Mobile Communication (GSM) and Digital Personal Communications System (DCS) handsets as emitted by handsets operating in the center of the communication band, that is, at 902 MHz (GSM) and 1747 MHz (DCS). Restrained mice were exposed for 2 h per day, 5 days per week over a period of 2 years to three different whole-body averaged specific absorption rate (SAR) levels of 0.4, 1.3, 4.0 mW/g bw (SAR), or were sham exposed. Regarding the organ-related tumor incidence, pairwise Fisher's test did not show any significant increase in the incidence of any particular tumor type in the RF exposed groups as compared to the sham exposed group. Interestingly, while the incidences of hepatocellular carcinomas were similar in EMF and sham exposed groups, in both studies the incidences of liver adenomas in males decreased with increasing dose levels; the incidences in the high dose groups were statistically significantly different from those in the sham exposed groups. Comparison to published tumor rates in untreated mice revealed that the observed tumor rates were within the range of historical control data. In conclusion, the present study produced no evidence that the exposure of male and female B6C3F1 mice to wireless GSM and DCS radio frequency signals at a whole body absorption rate of up to 4.0 W/kg resulted in any adverse health effect or had any cumulative influence on the incidence or severity of neoplastic and non-neoplastic background lesions, and thus the study did not provide any evidence of RF possessing a carcinogenic potential.