Effects of soft X-ray radiation damage on paraffin-embedded rat tissues supported on ultralene: a chemical perspective.

Radiation damage is an important aspect to be considered when analysing biological samples with X-ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X-ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation-damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X-ray exposure at different doses on ultralene, paraffin and on paraffin-embedded rat tissues were studied using Fourier-transform infrared (FTIR) microspectroscopy and X-ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose-dependent degradation of their vibrational profiles, consistent with radiation-induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.

A combination of stereological methods, biochemistry and electron microscopy for the investigation of drug treatment effects in experimental animals.

Some chemotherapeutic agents used for breast cancer (BC) treatment can induce severe side effects in the ovarian tissue. The combination of cyclophosphamide and docetaxel (TC) is widely used for BC treatment; however, its late effects in the ovary are not completely understood. The main purpose of this study was to evaluate the structural and ultrastructural alterations in the ovarian stroma induced by TC treatment. Wistar rats were divided into two groups: a control group and a TC group. They were euthanized 5 months after the end of treatment, and their plasma and ovaries were collected. Important alterations were noted. The serum estradiol level was significantly reduced in the TC group compared with the control group. Additionally, the number of apoptotic nuclei was higher in the TC group. The role of the inflammatory response in the development of ovarian damage was investigated, and we found an increased number of mast cells and increased expression of TNF-α in the TC group. The involvement of fibrosis was also investigated. The results showed that the TC group had increased expression levels of TGF-ß1, collagen type I (col-I) and collagen type III (col-III) compared with the control group. Ultrastructural analysis revealed the presence of collagen fibrils in the treated group and illustrated that the ovarian tissue architecture was more disorganized in this group than in the control group. The results from this study are important in the study of chemotherapy-induced ovarian failure and provide further insight into the mechanisms involved in the development of this disease.

On methods for radiometric surveying in radiotherapy bunkers.

Radiometric surveys in radiotherapy bunkers have been carried out in Brazil for many years, both by the same radiotherapy facility for verification of shielding as by the regulatory agency for licensing and control purposes. In recent years, the Intensity Modulated Radiation Therapy (IMRT) technique has been gradually incorporated into many facilities. Therefore, it has been necessary to consider the increased leakage component that has an important impact on the secondary walls. For that, a radiometric survey method has been used that considers an increased 'time of beam-on' for the secondary walls. In this work we discuss two methods of doing this: the first considers that this 'time of beam-on' affects the sum of the two components, leakage and scattered. In another method it is considered that only the leakage component is affected by this extended 'time of beam-on'. We compare the methods and show that for secondary walls withU= 1 the first method overestimates dose rates by important percentages and for secondary walls withU< 1 it can both overestimate or underestimate the dose rates, depending on the parameters of the project. An optimized procedure is proposed, according to the use factor (U) of the secondary wall to be measured.

Caspase-3 activation and increased procollagen type I in irradiated hearts.

The caspase-3-cleaved presence was evaluated in this study in the heart of irradiated rats, during the decline of ventricular function. Female Wistar rats were irradiated with a single dose of radiation (15 Gy) delivered directly to the heart and the molecular, histological and physiological evaluations were performed at thirteen months post-irradiation. The expressions of procollagen type I, TGF-ß1 and caspase-3-cleaved were analyzed using Western blotting. Cardiac structural and functional alterations were investigated by echocardiography and electron microscopy. In the irradiated group, the levels of procollagen type I, TGF-ß1 and caspase-3-cleaved are increased. Significant histological changes (degeneration of heart tissue and collagen deposition) and functional (reduced ejection fraction) were observed. Data suggest that the cardiac function decline after exposure to ionizing radiation is related, in part, to increased collagen and increased caspase-3-cleaved.

Determination of the correction factors used in Fricke dosimetry for HDR 192Ir sources employing the Monte Carlo method.

PURPOSE: Fricke dosimetry has shown great potential in the direct measurement of the absolute absorbed dose for 192Ir sources used in HDR brachytherapy. This work describes the determination of the correction factors necessary to convert the absorbed dose in the Fricke solution to the absorbed dose to water. METHODS: The experimental setup for Fricke irradiation using a 192Ir source was simulated. The holder geometry used for the Fricke solution irradiation was modelled for MC simulation, using the PENELOPE. RESULTS: The values of the factors determined for validation purposes demonstrated differences of less than 0.2% when compared to the published values. Four factors were calculated to correct: the differences in the density of the solution (1.0004 ± 0.0004); the perturbations caused by the holder (0.9989 ± 0.0004); the source anisotropy and the water attenuation effects (1.0327 ± 0.0012); and the distance from the center of the detection volume to the source (7.1932 ± 0.0065). CONCLUSION: Calculated corrections in this work show that the largest correction comes from the inverse squared reduction of the dose due to the point of measurement shift from the reference position of 1 cm. This situation also causes the correction due to volume averaging and attenuation in water to be significant. Future versions of the holder will aim to reduce these effects by having a position of measurement closer to the reference point thus requiring smaller corrections.

A pilot study of a postal dosimetry system using the Fricke dosimeter for research irradiators.

Cobalt-60 irradiators and soft X-ray machines are frequently used for research purposes, but the dosimetry is not always performed using the recommended protocols. This may lead to confusing and untrustworthy results within the conducted research. Postal dosimetry systems have already been approved by the IAEA, with thermoluminescence dosimeters (TLD) and optically stimulated luminescence (OSL) as the most commonly used dosimeter systems in these cases. The present study tests the Fricke dosimeter properties as a potential system to be used in postal dosimetry for a project using research irradiators. The Fricke solution was prepared according to the literature, and the linearity and fading tests were performed accordingly. All calculated doses were measured using a NE2571 Farmer ionization chamber as a reference. Doses ranging from 25 to 300 Gy were delivered by a research irradiator, with 150 kV and 22 mA to the Fricke solutions inside polyethylene (PE) bags (4 × 4 × 0.2 cm3). The results compared with the ionization chamber showed a linear response to the range of doses used. Fading tests showed no significant difference for the absorbed doses over 9 days, with a maximum difference of 1.5% found between days 0 and 3. The Fricke dosimeter presented good linearity, for low and high doses, and low uncertainties for the fading even for 9 days after irradiation. These preliminary results are motivating, and as the next step, we intend to design a postal dosimetry system using the PE bags of Fricke solution.

Technical Note: Fricke dosimetry for blood irradiators.

PURPOSE: The Fricke dosimeter has been shown to be a viable option as an absorbed dose standard. This work aims to provide the dose distribution in an irradiator container during blood irradiation using Fricke dosimetry. METHODS: Measurements were performed using a Gammacell Elan 3000 blood irradiator at Hemocenter in Rio de Janeiro, Brazil. A specific phantom was constructed and patented by the authors to perform these measurements. Fricke solution was prepared according to international protocols, and polyethylene bags filled with Fricke solution (n = 19) were spatially distributed within the phantom. Control bags were also submitted to the same process, except the irradiation. The irradiation time was calculated to give 25.7 Gy to the central portion of the phantom, the same dose used for blood bags. RESULTS: Encouraging results were obtained with an overall uncertainty of 2.1% (k = 1). The obtained results were compared with the doses calculated by the physicist from Hemocenter based on parameters provided by the manufacturer. The mean dose delivered to the Fricke bag in the center of the phantom (cavity 2) was 28.7 ± 0.5 Gy, which is 12% higher than the planned dose of 25.7 Gy. CONCLUSIONS: The obtained results showed that the setup (Fricke and phantom) is able to perform dosimetry for blood irradiators. The delivered dose was higher than expected. This highlights the importance in controlling all the parameters during irradiation to ensure the correct dose for all irradiated bags.

Biological effects induced by doses of mammographic screening.

PURPOSE: Mammography is the diagnostic imaging practice used in screening to detect early lesions suspected of malignancy. It uses a low energy X-ray beam in which a low dose in the order of 2-3 mGy is delivered to patient breast cells. However, it has been speculated that it could lead to significant cell damage, when compared to conventional X-ray. We investigated the biological effects of low doses, with mean glandular doses (MGDs) of 2.5 mGy and 2.5 + 2.5 mGy, on mammary cells in vitro. METHODS: We used the non-tumorigenic cell line (MCF-10A) and two tumor cells lines (MCF-7 and MDA-MB-231). Colony formation, apoptosis, and double-strand DNA breaks (DSBs) were quantified. RESULTS: The selected MGD regimens did not alter the formation of colonies by any of the cell lines. MCF-7 cells exhibited a markedly increase in apoptosis, 24 h after the single-dose protocol; MCF-10A cells underwent apoptosis only after 72 h, with both irradiation regimens, while MDA-MB-231 cells (highly invasive and metastatic) were not susceptible to apoptosis. The detection of γH2AX histone in the nuclei of irradiated cells showed that the double-dose resulted in increase of DSBs, especially in tumor cell lines. CONCLUSIONS: Although the health benefits of early breast screening remain indisputable, our future perspective is to better understand the biological basis for the effects of low dose radiation on breast cells and to investigate if and under what conditions there would be a risky situation in repeated mammography screening, in both asymptomatic and symptomatic women.

Preliminary pre-clinical studies on the side effects of breast cancer treatment.

Purpose: Technological advancement in the treatment of cancer together with early detection and diagnosis have considerably improved the survival of breast cancer patients. On the other hand, the potential of patients developing side effects from cancer treatment are not negligible. Despite the progress that has been made in terms of early diagnosis, therapy, and survival, including improvements in the chemotherapeutic agents, radiation and molecular targeted therapies, cardiotoxicity of cancer therapy is still cause for concern. Radiation therapy for breast cancer is associated with increased risk of heart disease and myocardial infarction. Furthermore, the association of radiation therapy to chemotherapy is an important aspect to be considered in the development of cardiac disease, as this could play an additional role as a risk factor. Besides the heart effect, other side effects can be observed in the bone, ovary, uteri, and other organs. This paper aims to review the recent literature to present the current understanding of side effects associated with breast cancer treatment. The focus is on recent preclinical studies that have assessed potential changes in different organs that may be injured after breast cancer treatment, both due to both radiation and chemotherapy agents.Conclusion: Radiation-induced heart disease is one important side effect that must be considered during the treatment planning and patient follow-up. The cardiac damage can be potentialized when chemotherapy is associated to radiotherapy, and the literature findings indicate that heart fibrosis plays an important role at the radio-chemotherapy induced cardiac damage. Literature findings also showed important side effects at the bone, that can lead to ospeoporosis, due to the decrease of calcium, after radio or chemotherapy treatments. This decrease could be explained by the ovarian failure observed at rats after chemotherapy treatment. It is of great importance to acknowledge the complications originating from the treatment, so that new strategies can be developed. In this way, it will be possible to minimize side effects and improve the patients' quality of life.

Feasibility study of the Fricke chemical dosimeter as an independent dosimetric system for the small animal radiation research platform (SARRP).

For the small animal radiation research platform (SARRP) with X-ray beams in the medium energy range (tube operating voltage at 220 kVp), reference dosimetry is based on the AAPM TG-61 recommendations following the in-phantom method. The objective of this study was to evaluate the feasibility of the Fricke solution as a dosimeter to determine the absorbed dose to water. Feasibility studies at this X-ray energy range are not widely available. We evaluated the accuracy, dose linearity and dose rate dependence in a comparison with an NE 2571 Farmer ionization chamber (IC) and measurements in water. The G(Fe3+) factor was calculated from the curve fitting of the chemical yields for two radioactive sources (192Ir and 60Co) and one X-ray system with a tube operating at 150 and 250 kVp. The same methodology was followed for the dependence of the G(Fe3+) value on the energy and the dose agreement assessment for 180 and 200 kVp in the SARRP. The Fricke system exhibits a good linear response over the range of 5-70 Gy and an accuracy better than 2% for a 2 Gy/min dose rate. The dose rate dependence is smaller than 1% for dose rates greater than 1 Gy/min. The dependence of the G(Fe3+) value on the energy is smaller than 0.41%, with dose agreements better than 2%. The feasibility of the dosimeter for measurements at high doses and high dose rates makes it a suitable tool for dosimetric verifications in several preclinical irradiation configurations.

Determination of the absorbed dose to water for medium-energy x-ray beams using Fricke dosimetry.

PURPOSE: For x-ray beams in the low and medium energy range, reference dosimetry is established in terms of air kerma. Fricke dosimetry has shown great potential in the absolute measurements of the absorbed dose to water for high-energy ranges. Therefore, the main purpose of this work was to compare the absorbed dose to water for medium-energy x-ray beams obtained through Fricke dosimetry with that obtained from the air kerma rate. METHODS: To determine the absorbed dose to water using Fricke dosimetry, the polyethylene bags methodology was chosen. Fricke solution was irradiated at four different beam qualities. The absorbed dose to water values obtained using Fricke dosimetry were compared to those obtained using the standard protocol, using the Z-score. RESULTS: Values of the Z-score were <2 for all measurements of absorbed dose to water, which means that the values obtained using Fricke dosimetry are equivalent to those obtained using the reference protocol. The combined standard uncertainty for the absorbed dose to water obtained by Fricke dosimetry was lower than that obtained with the ionization chamber. CONCLUSIONS: Chemical dosimetry using a standard FeSO4 solution has been demonstrated to be a potential option as a standard for the quantity absorbed dose to water for medium kV x-ray qualities.

Brazilian regulatory authority contribution to the shielding dimensioning model of radiotherapy rooms proposed by the NCRP 151.

The National Council on Radiation Protection and Measurements (NCRP) Report No. 151 is an essential document for bunker design commonly applied for radiotherapy treatment rooms. This document is used as a reference by several countries, including Brazil. The objective of this study is to evaluate the shielding dimensioning methodology recommended by NCRP 151, and compare it with the one adopted by the Brazilian regulatory authority. Radiotherapy rooms and respective doors were designed to use linear accelerators operating at 6, 10, 15, and 18 MeV under two different ways: (a) applying exclusively the methodology recommended by the NCRP 151, and (b) taking into consideration the complementary recommendations from the Brazilian authorities. The results suggest that designers in Brazil can count on at least 4 and 11% safety margin for dimensioning primary barriers in controlled and free areas respectively. Also 8% for secondary barriers in controlled areas, 9.7% for secondary barriers adjacent to the primary belt of free areas, and 6.6% for the lead of the doors.

Aliskiren Reduces the Adrenal Zona Glomerulosa Apoptosis and Autophagy in Wistar Rats with 2K1C Hypertension.

Hypertension is a disease classified as primary or secondary, manifested not only by elevation of blood pressure but also involved in structural and functional changes of target organs. Renal artery stenosis is a leading factor of secondary hypertension, and its progress is associated with overactivation of the renin-angiotensin-aldosterone system (RAAS). Aliskiren is a renin inhibiting drug that suppresses RAAS and culminates in decreased renin release, plasma angiotensin II concentration, and inhibition of aldosterone secretion. In this sense, the aim of the present study was to analyze the structural and ultrastructural morphophysiology of the adrenal glomerular zone, after treatment with aliskiren in Wistar rats with 2K1C hypertension. Parameters as structure and ultrastructure of the adrenal glomerular zone, cellular apoptosis, nuclear cell proliferation, and AT1 receptor expression were analyzed by immunostaining and electron microscopy. Our results showed that the hypertensive animals treated with aliskiren presented a reestablishment of AT1 receptor expression and decrease in apoptosis and autophagy. In addition, treatment with aliskiren improves the cell aspects in the adrenal glomerular zone, evidenced by ultrastructural analysis through preserved nuclei and well-developed mitochondria. Therefore, our evidence suggests that aliskiren has a beneficial effect on the adrenal glomerular zone remodeling in animals with renovascular hypertension.

Validating Fricke dosimetry for the measurement of absorbed dose to water for HDR 192Ir brachytherapy: a comparison between primary standards of the LCR, Brazil, and the NRC, Canada.

Two Fricke-based absorbed dose to water standards for HDR Ir-192 dosimetry, developed independently by the LCR in Brazil and the NRC in Canada have been compared. The agreement in the determination of the dose rate from a HDR Ir-192 source at 1 cm in a water phantom was found to be within the k = 1 combined measurement uncertainties of the two standards: D NRC/D LCR = 1.011, standard uncertainty = 2.2%. The dose-based standards also agreed within the uncertainties with the manufacturer's stated dose rate value, which is traceable to a national standard of air kerma. A number of possible influence quantities were investigated, including the specific method for producing the ferrous-sulphate Fricke solution, the geometry of the holder, and the Monte Carlo code used to determine correction factors. The comparison highlighted the lack of data on the determination of G(Fe3+) in this energy range and the possibilities for further development of the holders used to contain the Fricke solution. The comparison also confirmed the suitability of Fricke dosimetry for Ir-192 primary standard dose rate determinations at therapy dose levels.

Radiotherapy-Induced Skin Reactions Induce Fibrosis Mediated by TGF-ß1 Cytokine.

PURPOSE: This study aimed to investigate radiation-induced lesions on the skin in an experimental animal model. Methods and Materials: Cutaneous wounds were induced in Wistar rats by 4 MeV energy electron beam irradiation, using a dose rate of 240 cGy/min, for 3 different doses (10 Gy, 40 Gy, and 60 Gy). The skin was observed 5, 10, and 25 days (D) after ionizing radiation exposition. RESULTS: Infiltrate inflammatory process was observed in D5 and D10, for the 40 Gy and 60 Gy groups, and a progressive increase of transforming growth factor ß1 is associated with this process. It could also be noted a mischaracterization of collagen fibers at the high-dose groups. CONCLUSION: It was observed that the lesions caused by ionizing radiation in rats were very similar to radiodermatitis in patients under radiotherapy treatment. ADVANCES IN KNOWLEDGE: This study is important to develop strategies to prevent radiation-induced skin reactions.

Synchrotron microtomography to evaluate effects of different polychemotherapy drugs on cortical bone structure.

PURPOSE: To determine the effects of different polychemotherapy drugs on cortical bone structure, the femur diaphysis of rats were treated with two different chemotherapy drugs, AC (doxorubicin + cyclophosphamide) and TC (docetaxel + cyclophosphamide), and evaluated by 3D morphological analysis using synchrotron radiation microtomography. MATERIALS AND METHODS: Wistar rats were classified into three groups. One group received doses of docetaxel and cyclophosphamide (G1) - TC regimen; a second group received doses of doxorubicin and cyclophosphamide (G2) - AC regimen; while a control group (G0) received no further treatment. 3D tomographic images of the rats' femurs were obtained at the SYRMEP (Synchrotron Radiation for Medical Physics) beamline at the ELETTRA Synchrotron Laboratory in Trieste, Italy, using monochromatic X-rays with resolution of 9 µm. RESULTS: It could be shown that the treatment caused significant differences in morphological parameters measured from the 3D images of femur diaphysis of rats, among the studied groups, complementing a previous study using stereological methods, biochemistry and electron microscopy. CONCLUSIONS: Our results indicate that the same process of osteoporosis caused by advancing age might occur in young women treated with docetaxel + cyclophosphamide (TC) and doxorubicin + cyclophosphamide (AC). 3D microtomography was shown to be an outstanding technique for bone analysis.

A feasibility study of Fricke dosimetry as an absorbed dose to water standard for 192Ir HDR sources.

High dose rate brachytherapy (HDR) using 192Ir sources is well accepted as an important treatment option and thus requires an accurate dosimetry standard. However, a dosimetry standard for the direct measurement of the absolute dose to water for this particular source type is currently not available. An improved standard for the absorbed dose to water based on Fricke dosimetry of HDR 192Ir brachytherapy sources is presented in this study. The main goal of this paper is to demonstrate the potential usefulness of the Fricke dosimetry technique for the standardization of the quantity absorbed dose to water for 192Ir sources. A molded, double-walled, spherical vessel for water containing the Fricke solution was constructed based on the Fricke system. The authors measured the absorbed dose to water and compared it with the doses calculated using the AAPM TG-43 report. The overall combined uncertainty associated with the measurements using Fricke dosimetry was 1.4% for k = 1, which is better than the uncertainties reported in previous studies. These results are promising; hence, the use of Fricke dosimetry to measure the absorbed dose to water as a standard for HDR 192Ir may be possible in the future.

Apoptosis induction of cardiomyocytes and subsequent fibrosis after irradiation and neoadjuvant chemotherapy.

PURPOSE: Breast cancer treatments can induce important cardiovascular complications. The aim of this study was to evaluate cardiac alterations after irradiation and chemotherapy in an animal model. MATERIAL AND METHODS: Wistar rats were divided into three groups: Control, TC+ IR (received chemotherapy and irradiation) and IR (received only irradiation). After 5 months, echocardiography was performed, the animals were euthanized, and the left ventricle was analyzed using light microscopy techniques and Polymerase Chain Reaction (PCR). RESULTS: Echocardiography showed decreases in ejection fraction and cardiac output, in TC+ IR group. Both TC+ IR and IR showed reduced intramyocardial vessel-to-cardiomyocyte ratio, increased connective tissue, cardiomyocyte hypertrophy, increased numbers of apoptotic nuclei and increased Bax/Bcl2 expression. We also observed increased Transforming growth factor (TGF) beta 1 mRNA expression in both groups, but type 1 Procollagen expression was increased in TC+ IR group only. CONCLUSIONS: The study suggests that the induced cardiac remodelling begins with the reduction of intramyocardial vessels in the left ventricle tissue. The main consequence is the loss of cardiomyocytes through apoptosis, leading to the replacement of healthy tissue by fibrous tissue. It was observed that the damage caused by the combination of irradiation and chemotherapy induced functional alterations that did not occur when the animals were only irradiated.

Chemotherapy and radiation regimens to breast cancer treatment induce changes in mRNA levels of renin-angiotensin system related genes in cardiac tissue.

BACKGROUND AND AIM: Cardiovascular complications are one limitation of breast cancer treatment. The aim of the current study was to investigate whether the renin-angiotensin related genes could be altered by chemotherapy and radiotherapy, using a rat model. METHODS: Female rats were divided into three groups: control, chemotherapy + irradiation (TC+IR) and irradiation (IR). Molecular analyses of the left ventricle were performed five months after the end of treatment. The analyses evaluated the changes in mRNA levels of some renin-angiotensin system (RAS) related genes: angiotensinogen, renin, angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) and vascular endothelial growth factor (VEGF), which can be related to ACE production, by RT-PCR. RESULTS: Renin was only observed in treated groups, TC+IR and IR, compared with the control group. ACE and VEGF levels were decreased in TC+IR (p<0.001) and in IR (p<0.001), and AT1 mRNA was higher in groups TC+IR (p<0.01) and IR (p<0.05) compared with the control group. CONCLUSION: Chemotherapy and irradiation can induce significant changes in some RAS related genes. These alterations are important to understand the pathways and consequences beyond cardiotoxicity induced by breast cancer treatments.

Evaluation of rib microstructure in Wistar rats using SR-µCT after radiation therapy simulation for breast cancer.

A better understanding of biological interactions that occur after exposure to photon radiation is needed in order to optimize therapeutic regimens and facilitate development and strategies that decrease radiation-induced side effects in humans. In this work, ribs of Wistar rat submitted to radiotherapy simulation were imaged using synchrotron radiation computed microtomography at Elettra Synchrotron Laboratory in Trieste, Italy. Histomorphometric parameters were calculated directly from the 3D microtomographic images and showed significant differences between irradiated and non-irradiated groups.