Elemental detection and mapping of rat bone matrix induced by chemoradiotherapy with confocal μ-XRF

Elemental detection and mapping of rat bone matrix induced by chemoradiotherapy with confocal μ-XRF | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Elemental detection and mapping of rat bone matrix induced by chemoradiotherapy with confocal μ-XRF Qiuxia Li, Hongchi Chen, Lazhen Zhou, Fangzuo Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4180548/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Breast cancer is among the most prevalent malignant tumors in women. The administration of chemotherapy and radiotherapy for breast cancer treatment can lead to amenorrhea and substantial bone loss in women. Consequently, postmenopausal women are susceptible to the morbidity and mortality risks associated with breast cancer and postmenopausal osteoporosis. In this study, we investigated the impact of chemotherapy and radiotherapy on osteoporosis in female rats using confocal microbeam X-ray fluorescence (µ-XRF) method. The female Sprague Dawley (SD) rats were categorized into three groups: the control group (G1), the chemotherapy and radiotherapy group (G2), and the radiotherapy-only group (G3). The SD rats were euthanized six weeks post chemotherapy and radiotherapy, and femur slices with a thickness of 1mm were obtained for confocal µ-XRF analysis. The results demonstrate a significant loss of calcium in the G2 and G3 groups, while the G2 group exhibited a substantial increase in Fe content compared to the G1 group. The conclusion can be drawn that the occurrence of osteoporosis is related to chemotherapy and radiotherapy, while the significant elevation in bone iron content signifies the progression of osteoporosis. Elemental analysis/imaging Confocal µ-XRF Bone matrix Chemotherapy Radiotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction In 2020, female breast cancer emerged as the predominant cause of global cancer incidence, surpassing lung cancer, with an estimated 2.3 million new cases accounting for 11.7% of all reported cancer cases [ 1 ]. Common treatment strategies for breast cancer include chemotherapy and radiotherapy [ 2 – 5 ]. The administration of treatments such as chemotherapy and radiotherapy can induce temporary or non-temporary amenorrhea in premenopausal women, thereby reducing estrogen production. This hormonal alteration may contribute to accelerated bone loss, the development of osteoporosis, and an increased risk of fractures [ 6 , 7 ]. Therefore, it is imperative to assess the impact of chemotherapy and radiotherapy on bone loss and osteoporosis. The measurement of bone mineral density (BMD) is widely recognized as the gold standard in clinical practice for assessing the risk of osteoporotic fractures. In addition to potassium (K), calcium (Ca), and phosphorus (P), bones also contain various mineral microelements, including iron (Fe), zinc (Zn), strontium (Sr), among others. The presence of microelements is indispensable for the normal growth and development of the skeletal system in both humans and animals. Despite their minor role as building components in bones, microelements play crucial functional roles in bone metabolism and turnover, making them significant factors influencing the development of osteoporosis [ 8 ]. For instance, Fe accumulation, serving as an independent risk factor for osteoporosis, can significantly expedite bone loss in postmenopausal women afflicted with osteoporosis [ 9 , 10 ]. Tsay et al. conducted a Fe intervention in mice and found that the mice with accumulated Fe experienced severe bone loss and a significant decrease in bone density [ 11 ]. Jia et al. replicated the Fe intervention study and also discovered that excessive iron leads to bone loss, which is associated with the activation of osteoclasts and an increase in oxidative stress levels [ 12 ]. Zn can increase bone mass by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption [ 13 – 15 ]. Sr can promote bone formation and inhibit bone resorption [ 16 – 18 ]. The traditional methods used for determining bone mineral microelements include flame atomic absorption spectrometry (FAAS), atomic fluorescence spectrometry (AFS), and inductively coupled plasma-based techniques (ICP) [ 19 – 21 ]. FAAS, AFS, and ICP are destructive methods, because they require the samples to undergo chemical pretreatment (i.e., dissolution or digestion) before analysis. Compared with the elemental analysis method mentioned above, the X-ray fluorescence (XRF) method can simply, quickly, and nondestructively obtain content and distribution information of elements in samples. XRF has been extensively employed in the biomedical field to elucidate the intrinsic correlation between analysis outcomes and alterations in the physiological milieu of diseases, particularly for ascertaining the content and distribution of trace elements within bone [ 22 – 31 ]. For instance, Gomez et al. employed synchrotron radiation X-ray fluorescence (SRXRF) to investigate the spatial distribution of zinc in human bone and pig osteogenesis, revealing a non-uniform distribution pattern with the highest concentration observed on the surface of haversian bone [ 22 ]. Moise et al. conducted a longitudinal study using in vivo XRF to monitor the long-term intake of self-supplementing strontium citrate in osteoporotic women, and observed a continuous increase in Sr levels even after 4 years, highlighting the necessity of monitoring prolonged usage of Sr supplements [ 23 , 24 ]. Fei et al. employed SRXRF to investigate alterations in femur elemental composition in a diabetic osteoporosis animal model [ 29 ]. Their findings revealed a significant reduction in femur bone density among diabetic mice, accompanied by notable decreases in the relative mineral contents of Ca, P, Sr, and Zn; however, no significant differences were observed for Cr, Fe, Cu, and Pb. The biological samples often exhibit a multitude of components, intricate structures, and non-uniform distribution. XRF analysis of the entire sample solely reflects the average composition, while investigation of biochemical reactions occurring in the microenvironment necessitates analysis at the tissue or even cellular level. By quantifying the micro-distribution and content variations of elements within bone tissue and/or bone cells, a more comprehensive understanding of aberrant bone metabolism can be attained. The confocal µ-XRF technique represents a significant advancement in XRF methodology, surpassing the conventional multielement analysis capabilities of traditional XRF methods. The confocal µ-XRF technique offers notable advantages including exceptional sensitivity, precise detection, and micro-area analysis, enabling the acquisition of three-dimensional elemental distributions [ 32 – 37 ]. In this study, our primary objective was to assess the impact of chemotherapy and radiotherapy on the femoral distribution of Ca, Fe, Zn, K, and Sr in rats using confocal µ-XRF analysis. Additionally, we aimed to investigate the potential association between these determined microelements and bone loss. 2. Materials and methods 2.1. Animals Fifteen 12-week-old female Sprague Dawley (SD) rats, weighing 200 g each, were utilized for this study. The experimental animals used in this study were sourced from the Experimental Animal Center of Gannan Medical University. The rats were housed in ventilated cages maintained at standard environmental conditions (25 ± 2 o C, 50% ± 5% humidity and a 12/12 h light/dark cycle) with ad libitum access to standard water and diet. The SD rats were randomly allocated into three groups (n = 5 for each group), designated as G1, G2, and G3, respectively. The control group consisted of SD rats in G1 group. SD rats in the G2 group were administered a single intraperitoneal injection of docetaxel (15 mg/kg) and cyclophosphamide (60 mg/kg) chemotherapy drugs on the first day of each week for a duration of 4 weeks [ 38 ]. On the 7th day of the final chemotherapy cycle (i.e., week 4), SD rats in the G2 group underwent thoracic radiotherapy that simulated breast cancer radiotherapy. The SD rats in the G3 group were not subjected to chemotherapy but instead received a single dose of 20 Gy radiation in the supine position using a linear accelerator Varian® (Clinac 2100). The X-ray beam had a nominal energy of 6 MV, and the dose rate was 240 cGy/min. The irradiation was administered to the left thoracic region of the rats within a 2 cm 2 field. The irradiation of SD rats with a dose of 20 Gy was found to be bioequivalent to the typically administered dose of 45 to 60 Gy in BC patients [ 39 ]. After irradiation, SD rats were returned to their cages and subjected to daily monitoring for survival, dietary intake, and physical activity. SD rats in the G1 and G3 groups were administered a single intraperitoneal injection of 0.9% normal saline, with an equivalent volume as that given to the G2 group, on the first day of each week for four consecutive weeks. The experimental procedures involving animals in this study were conducted in strict accordance with the guidelines for the management and utilization of experimental animals published by the National Scientific and Technological Academic Works Publishing Fund Committee. Additionally, all protocols were approved by both the Ethics Committee and Animal Experiment Committee of Gannan Medical University. 2.2. Sample preparation Referring to animal treatment in Ref. 29, the rats that had undergone fasting were humanely euthanized using pentobarbital anesthesia at the sixth week after the end of radiotherapy and chemotherapy. The right femurs in G1, G2, and G3 groups were promptly dissected and thoroughly cleared of any adherent soft tissue. The femurs were sectioned into 1 mm thick slices using a diamond blade and subsequently polished to achieve a flat surface. Confocal µ-XRF analysis was conducted on the cancellous portion of the specimens. 2.3. Confocal µ-XRF The elemental analysis of all femur samples was conducted using confocal µ-XRF, and a schematic diagram illustrating the scanning spectrometer is presented in Fig. 1 . The confocal µ-XRF spectrometer comprises an X-ray source, a polycapillary focusing X-ray lens (PFXRL), a polycapillary parallel X-ray lens (PPXRL), a sample stage, a charge-coupled device camera, an X-ray detector, a signal amplifier, a counting circuit, and a computer. The comprehensive elucidation of the confocal µ-XRF technique can be found in our previously published work [ 37 ]. The detector is capable of detecting only XRF signals originating from the confocal microvolume formed by the overlapping of the PFXRL output focal spot and the PPXRL input focal spot. By precisely manipulating the sample stage, the confocal microvolume can be dynamically relocated within the specimen, facilitating high-resolution elemental scanning analysis in situ on a point-to-point basis. The samples in this experiment were scanned point-by-point using a dwell time of 20 s, a scanning area of 3×3 mm 2 , and an effective step (pixel) size of 20 µm. The optical micrograph in Fig. 2 presents a representative view of the femur sample, accompanied by a confocal µ-XRF scanning area. The elemental concentration data from each pixel was collected and utilized to generate a corresponding distribution mapping of the elements. 2.4. Elemental analysis The excited XRF photons in the confocal microvolume are emitted isotropically. The relationship between the XRF intensity of element i detected and its concentration can be expressed as follows [ 40 ]: $${I}_{i}\propto {S}_{i}\bullet {C}_{i}\bullet A$$ 1 Here, I i represents the net intensity of the characteristic XRF peak for element i (counts per second, cps), C i denotes the concentration of elemen i (µg⋅g − 1 ), S i signifies the sensitivity factor for analyzing element i (cps⋅cm⋅g − 1 ), and A represents the absorption factor for element i . The confocal µ-XRF system was calibrated using a standard sample of NIST (National Institute of Standards and Technology, USA) bone meal to obtain the above experimental parameters. The inorganic components of bone are mainly Ca and P, and trace elements are K, Fe, Zn, Sr, etc. For the bone meal NIST SRM-1486, the certified Ca concentration is 26.58 ± 0.24%. In this study, about 100 mg of bone meal NIST SRM-1486 was pressed to obtain a tablet with a thickness of 1 mm. The sensitivity and minimum detection limits (MDL) for each element were determined by analyzing standard bone meal samples purchased from Shenzhen Deborui Biological Technology Co., LTD. The standard samples were placed on 6 µm thick Mylar membranes. The blank spectrum of the Mylar film was processed, and subsequently, the calculated concentrations of elements in the samples were adjusted by subtracting the corresponding blank values. The X-ray spectra were subjected to peak fitting using the AXIL program package in order to extract the net intensities of XRF peaks [ 41 ]. One of five femur samples in each group were randomly selected and were scanned by Confocal µ-XRF microprobe for element analysis. The data were removed if they were less than three times the corresponding standard deviation. The elemental peak area was normalized to the peak counts of Ar, which is present in air at a constant proportion. The normalized peak areas were utilized to estimate the relative elemental contents. The sample thickness, measurement geometry, and beam energy remained unchanged to ensure the consistent application of element-specific conversion factors across all measurements. 3. Results and discussion The MDL for element i can be expressed as [ 42 ]: $${m}_{MDL}=3\bullet {C}_{i}\bullet \frac{\sqrt{{I}_{B}}}{{I}_{i}}$$ 2 Here, I i represents the net fluorescent line intensity and I B denotes the background intensity of the considered element i . The MDL of the confocal µ-XRF spectrometer for the target elements can be found in Table 1 . Table 1 Minimum detection limit (MDL) for target elements (µg/g). Elements \({m}_{MDL}\) Ca 149 ± 10 K 14.01 ± 1.14 Fe 8.19 ± 1.03 Zn 4.33 ± 0.42 Sr 22.931 ± 2.44 Figure 3 shows a typical XRF spectrum of G2 femur measured by confocal µ-XRF method, which encompasses characteristic X-rays emitted by various elements present in the bone matrix. The XRF spectrum revealed the presence of several microelements, including S, K, Mn, Fe, Cu, Zn, Pb and Sr in the femur sample. This finding suggests that confocal µ-XRF can effectively and simultaneously detect a wide range of trace elements in the analyzed samples. The spatial distribution of K, Ca, Fe, Zn, and Sr within the scanning region obtained through confocal µ-XRF analysis is depicted in Figs. 4 to 6 . The visual analysis of Fig. 4 ~ 6 reveals that the distribution of Ca was relatively homogeneous, while the distributions of K, Fe, Zn, and Sr exhibited non-uniform patterns. Notably, a significant deposition of iron in bone matrix was observed specifically in the G2 group. The two-dimensional distribution of elements matches the morphological characteristics, which can reveal the elemental correlations of different femurs. The average relative element contents of the investigated elements in the scanning region of G1, G2 and G3 are given in Table 2 . Compared to the G1 group, significant calcium loss was observed in both the G2 and G3 groups. The average relative element contents reveal no significant differences in K, Zn, and Sr; however, notable disparities are observed in Fe. This suggests that chemotherapy induces overexpression of Fe, leading to competition with calcium ions. Fe serves as a cofactor in enzymes involved in the synthesis of collagen bone matrix, as well as in 25 OH vitamin D hydroxylase, an enzyme responsible for activating vitamin D and facilitating calcium absorption [ 43 , 44 ]. The objective of this study was to examine the impact of chemotherapy and radiotherapy on the elemental composition and distribution within the bone matrix. Relevant literature has demonstrated that radiotherapy and chemotherapy have the potential to induce alterations in bone mineral density [ 45 , 46 ]. For instance, Nogueira et al. conducted a micro-XRF analysis to assess the impact of photon irradiation treatment on calcium levels in the dorsal ribs of Wistar rats and observed a significant decrease in calcium content following radiotherapy [ 45 ]. Our radiotherapy findings were consistent with the results obtained by Nogueira et al. However, to the best of our knowledge, there is currently no assessment available regarding crucial microelements in the bone matrix after chemotherapy. The results of our study demonstrate the efficacy of confocal µ-XRF method in analyzing crucial major and trace elements, including Ca, Fe, Zn, K, and Sr within the bone matrix. Table 2 Relative element contents (µg/g) in femur of control and chemotherapy/radiotherapy rat. K Ca Fe Zn Sr G1 33.87 ± 0.83 251.13 ± 11.07 9.22 ± 1.48 114.78 ± 1.22 81.29 ± 1.71 G2 37.73 ± 0.87 113.20 ± 11.05 25.35 ± 1.45 96.76 ± 1.24 100.73 ± 1.67 G3 25.71 ± 0.89 121.31 ± 11.09 13.63 ± 1.67 107.83 ± 1.17 91.72 ± 1.78 Postmenopausal osteoporosis can be considered as a biochemical phenomenon characterized by the presence of coarse and shortened bone mass, along with weakened microstructure resulting from the loss of estrogen’s direct impact on osteoclasts [ 47 ]. Therefore, the biochemical association between breast cancer and postmenopausal osteoporosis is a matter of significant concern and scientific interest. Both bone and breast tissue rely on estrogen, a pivotal hormone that regulates bone density and maintains a delicate equilibrium between bone formation and resorption by modulating osteoclast levels or promoting osteoblast proliferation [ 48 – 52 ]. Chemotherapy and radiotherapy-induced ovarian failure results in a decline in estrogen levels, subsequently leading to significant calcium loss and an elevation in bone iron content. The enhanced survival rates of breast cancer patients undergoing chemotherapy and radiotherapy underscore the significance of comprehending the mechanisms and long-term impacts of chemotherapy and radiotherapy-induced alterations in bone composition, encompassing both major and trace elements. The specific mechanism of Fe accumulation leading to postmenopausal osteoporosis remains unclear. The cellular-level studies have demonstrated that the accumulation of Fe can impede the activity and functionality of osteoblasts, stimulate osteoclast function, induce apoptosis in mesenchymal stem cells, and hinder their differentiation into osteoblasts [ 53 , 54 ]. It should be noted that the spatial resolution achieved with the confocal µ-XRF method is limited, which precludes studying structures at the (sub-) cellular level. In future investigations, our aim is to employ nano-XRF techniques for the detection of Fe distribution within osteoblasts and elucidate the underlying cellular and/or molecular mechanisms. This will provide profound insights into the impact of Fe metabolism on osteoporosis following breast cancer chemotherapy. 4. Conclusion The present study demonstrates the application of confocal µ-XRF as an analytical method for assessing the impact of chemotherapy and radiotherapy on alterations in content and distribution patterns of essential major and trace elements within bone matrix. The results suggest a significant decrease in Ca levels following chemotherapy and radiotherapy, indicating the presence of osteoporosis. In addition, chemotherapy results in a significant accumulation of iron in bone matrix. In conclusion, confocal µ-XRF has emerged as a robust analytical method for assessing the impact of chemotherapy and radiotherapy on osteoporosis. Chemotherapeutic drugs such as cyclophosphamide and docetaxel induce ovarian changes that result in reduced or insufficient estrogen levels, subsequently leading to alterations in bone matrix composition and the promotion of osteoporosis. The primary constraint of the proposed confocal µ-XRF method lies in its current limitation to preclinical testing exclusively in rats, rendering it ethically unfeasible for human experimentation and thereby diminishing its clinical applicability for population-wide screening and treatment. Declarations CRediT authorship contribution statement Qiuxia Li : Conceptualization, Methodology, Validation, Investigation, Formal analysis, Data curation, Writing-original draft. Hongchi Chen : Writing-review & editing. Lazhen Zhou : Writing-review & editing. Fangzuo Li : Conceptualization, Writing-review & editing, Supervision, Funding acquisition, Project administration. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgement This research was funded by the Natural Science Foundation of Jiangxi Province (Grant No. 20224BAB201020), and the Startup Fund of High-level Talents Scientific Research of Gannan Medical University (QD201805). References Sung H, Ferlay J, Siegel RL et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209-249 Kim DY, Youn JC, Park MS et al (2019) Cardiovascular outcome of breast cancer patients with concomitant radiotherapy and chemotherapy: A 10-year multicenter cohort study. J Cardiol 74:175-181 Kozak MM, Eyben RV, Gutkin PM et al (2021) Locoregional and Distant Outcomes in Women With cT1-3N1 Breast Cancer Treated With Neoadjuvant Chemotherapy With or Without Adjuvant Radiotherapy. Clin Breast Cancer 21:373-382 Di L, Liu LJ, Yan YM et al (2019) Discovery of a natural small molecule compound that suppresses tumor EMT, stemness and metastasis by inhibiting TGFβ/BMP signaling in triple-negative breast cancer. J Exp Clin Cancer Res 38:134 Zhang L, Yang F, Huang L et al (2017) Luteolin enhances the antitumor activity of lapatinib in human breast cancer cells. Biomed Res 28:4902-4907 Stumpf U, Kostev K, Siebenbürger G et al (2020) Influence of chemotherapy and endocrine treatment on fractures in postmenopausal women with breast cancer-a retrospective cohort study. J Bone Oncol 22:100292 Vestergaard P (2019) Drugs Causing Bone Loss, Handbook of experimental pharmacology. Zofkova I, Nemcikova P, Matucha P (2013) Trace elements and bone health. Clin Chem Lab Med 51:1555-1561 Zheng J, Mao X, Ling J et al (2014) Low serum levels of zinc, copper, and iron as risk factors for osteoporosis: a meta-analysis. Biol Trace Elem Res 160:15-23 Weinberg ED (2006) Iron loading: a risk factor for osteoporosis. Biometals 19:633-635 Tsay J, Yang Z, Ross FP et al (2010) Bone loss caused by iron overload in a murine model: importance of oxidative stress. Blood 116:2582-2589 Jia P, Xu YJ, Zhang ZL et al (2012) Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species. J Orthop Res 30:1843-1852 Yamaguchi M, Goto M, Uchiyama S et al (2008) Effect of zinc on gene expression in osteoblastic MC3T3-E1 cells: enhancement of Runx2, OPG, and regucalcin mRNA expressions. Mol Cell Biochem 312:157-166 Hie M, Iitsuka N, Otsuka T et al (2011) Zinc deficiency decreases osteoblasts and osteoclasts associated with the reduced expression of Runx2 and RANK. Bone 49:1152-1159 Yamaguchi M, Segawa Y, Shimokawa N et al (1992) Inhibitory effect of beta-alanyl-L-histidinato zinc on bone resorption in tissue culture. Pharmacology 45:292-300 Bonnelye E, Chabadel A, Saltel F et al (2008) Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. Bone 42:129-138 Marie PJ, Felsenberg D, Brandi ML (2011) How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis. Osteoporosis Int 22:1659-1667 Marx D, Yazdi AR, Papini M et al (2020) A review of the latest insights into the mechanism of action of strontium in bone. Bone Rep 12:100273 Louis O, Winke PVD, Covens P et al (1994) Mineral content of vertebral trabecular bone: Accuracy of dual energy quantitative computed tomography evaluated against neutron activation analysis and flame atomic absorption spectrometry. Bone 15:35-39 Ščančar J, Milačič R, Benedik M et al (2000) Determination of trace elements and calcium in bone of the human iliac crest by atomic absorption spectrometry. Clin Chim Acta 293:187-197 Mahanti HS, Barnes RM (1983) Determination of major, minor and trace elements in bone by inductively-coupled plasma emission spectrometry. Anal Chim Acta 151:409-417 Gomez S, Rizzo R, Mucelli MP et al (1999) Zinc Mapping in Bone Tissues by Histochemistry and Synchrotron Radiation-Induced X-Ray Emission: Correlation With the Distribution of Alkaline Phosphatase. Bone 25:33-38 Moise H, Adachi JD, Chettle DR et al (2012) Monitoring bone strontium levels of an osteoporotic subject due to self-administration of strontium citrate with a novel diagnostic tool, in vivo XRF: A case study. Bone 51:93-97 Moise H, Chettle DR, Milić AP (2014) Monitoring bone strontium intake in osteoporotic females self-supplementing with strontium citrate with a novel in-vivo X-ray fluorescence based diagnostic tool. Bone 61:48-54 Wohl GR, Chettle DR, Milić AP et al (2013) Accumulation of bone strontium measured by in vivo XRF in rats supplemented with strontium citrate and strontium ranelate. Bone 52:63-69 Sales E, Silva CERD, Letichevsky S et al (2020) Study of composition and structure of demineralized bone using X-ray techniques. Radiat Phys Chem 167:108310 Zhang XX, Specht AJ, Wells E et al (2021) Evaluation of a portable XRF device for in vivo quantification of lead in bone among a US population. Sci Total Environ 753:142351 Sánchez HJ, Valentinuzzi MC, Grenón M et al (2008) Total reflection X-ray fluorescence analysis of oral fluids of women affected by osteoporosis and osteopenia. Spectrochim Acta B 63:1485-1488 Fei YR, Zhang M, Li M et al (2007) Element analysis in femur of diabetic osteoporosis model by SRXRF microprobe. Micron 38:637-642 Ballarre J, Desimone PM, Chorro M et al (2013) Bone quality around bioactive silica-based coated stainless steel implants: Analysis by Micro-Raman, XRF and XAS techniques. J Struct Biol 184:164-172 Hahn M, Buzanich G, Jähn K et al (2019) Analysis of cobalt deposition in periprosthetic bone specimens by high-resolution synchroton XRF in undecalcified histological thin sections. Materialia 6:100290 Mazel V, Reiche I, Busignies V et al (2011) Confocal micro-X-ray fluorescence analysis as a new tool for the non-destructive study of the elemental distributions in pharmaceutical tablets. Talanta 85:556-561 Nakazawa T, Tsuji K (2013) Depth-selective elemental imaging of micro SD card by confocal micro XRF analysis. X-Ray Spectro 42:123-127 Lühl L, Mantouvalou I, Schaumann I et al (2013) Three-dimensional chemical mapping with a confocal XRF setup. Anal Chem 85:3682-3689 Nakano K, Nishi C, Otsuki K et al (2011) Depth elemental imaging of forensic samples by confocal micro-XRF method. Anal Chem 83:3477-3483 Yi L, Qin M, Wang K et al (2016) The three-dimensional elemental distribution based on the surface topography by confocal 3D-XRF analysis. Appl Phys A 122:856-862 Xu Q, Xia W, Zhou L et al (2022) Determination of hepatic iron deposition in drug-induced liver fibrosis in rats by confocal micro-XRF spectrometry. ACS Omega 7:3738-3745 Konat GW, Kraszpulski M, James I et al (2008) Cognitive Dysfunction Induced by Chronic Administration of Common Cancer Chemotherapeu-tics in Rats. Metab Brain Dis 23:325-333 Krüse JJ, Zurcher C, Strootman EG et al (2011) Structural changes in the auricles of the rat heart after local ionizing irradiation. Radiother Oncol 58:303-311 Bertin EP (1975) Principles and Practice of X-ray Spectrometric Analysis, Secondy. Plenun Press, London Bernasconi G, Tajani A, Kregsamer P (2000) Manual for QXAS/AXIL. Versão 3.5. International Atomic Energy Agency, Vienna Samber BD, Silversmit G, Schamphelaere KD et al (2010) Element-to-tissue correlation in biological samples determined by three-dimensional x-ray imaging methods. J Anal At Spectrom 25:544-553 D’Amelio P, Cristofaro MA, Tamone C et al (2008) Role of iron metabolism and oxidative damage in postmenopausal bone loss. Bone 43:1010-1015 Sato K, Nohtomi K, Demura H et al (1997) Saccharated ferric oxide (SFO)-induced osteomalacia: in vitro inhibition by SFO of bone formation and 1,25-dihydroxy-vitamin D production in renal tubules. Bone 21:57-64 Nogueira LP, Barroso RC, Almeida APD et al (2012) Evaluation of Photon Irradiation Treatment Upon Calcium Content of Ribs of Wistar Rats Using Micro-XRF. AIP Conference Proceedings 1437:104-107. Rusz O, Kahán Z (2013) Bone Homeostasis and Breast Cancer: Implications for Complex Therapy and the Maintenance of Bone Integrity. Pathol Oncol Res 19:1-10 Muhammad A, Mada SB, Malami I et al (2018) Postmenopausal osteoporosis and breast cancer: The biochemical links and beneficial effects of functional foods. Biomed Pharmacother 107:571-582. Trémollieres FA (2014) Screening for osteoporosis after breast cancer: For whom, why and when. Maturitas 79:343-348 Cauley JA (2015) Estrogen and bone health in men and women. Steroids 99:11-15 Manolagas SC (2010) From Estrogen-Centric to Aging and Oxidative Stress: A Revised Perspective of the Pathogenesis of Osteoporosis. Endocr Rev 31:266-300 Khalid AB, Krum SA (2016) Estrogen receptors alpha and beta in bone. Bone 87:130-135 Eastell R, Szulc P (2017) Use of bone turnover markers in postmenopausal osteoporosis. Lancet Diabetes Endo 5:908-923 Yang Q, Jian JL, Abramson SB, et al (2011) Inhibitory effects of iron on bone morphogenetic protein 2-induced osteoblastogenesis. J Bone Miner Res 26:1188-1196 Balogh E, Tolnai E, BélaNagy J, et al (2016) Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin. BBA-Mol Basis Dis 1862:1640-1649 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 21 May, 2024 Editor assigned by journal 05 Apr, 2024 Submission checks completed at journal 29 Mar, 2024 First submitted to journal 28 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4180548","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287787626,"identity":"b1b8f738-a484-4242-8c87-c65d612d2ea8","order_by":0,"name":"Qiuxia Li","email":"","orcid":"","institution":"Gannan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiuxia","middleName":"","lastName":"Li","suffix":""},{"id":287787627,"identity":"fc4db105-e7d6-45fa-a4d5-eb146422f781","order_by":1,"name":"Hongchi Chen","email":"","orcid":"","institution":"Gannan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongchi","middleName":"","lastName":"Chen","suffix":""},{"id":287787628,"identity":"9567d4c8-e469-45f2-b6e5-027628d13957","order_by":2,"name":"Lazhen Zhou","email":"","orcid":"","institution":"Gannan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lazhen","middleName":"","lastName":"Zhou","suffix":""},{"id":287787629,"identity":"5db7c7cd-3f5c-4be5-bc6f-411168513825","order_by":3,"name":"Fangzuo Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACPuYDjA8+GNjIsbE3Nj78QIwWNrYEZsMZFWnG/DyHm40liNTCJs1z5nDizBnpbQI8xGlhf2w4s+0w44abD9sYJBjs5HQbCGphSHzwsS2d2eB2YtuDAoZkY7MDhLTINxwG2mLNBtTSbiDBcCBxG0EtbIxt0rxtzDwGNw+2SfAQp4UZ5H1nCckZjERrYQMHsgE/TyIwkA2I8As/G/tDUFTWt7Eff/jwQ4WdHEEtaMCANOWjYBSMglEwCnAAAG1VQAKQZTX5AAAAAElFTkSuQmCC","orcid":"","institution":"Gannan Medical University","correspondingAuthor":true,"prefix":"","firstName":"Fangzuo","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-03-28 08:07:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4180548/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4180548/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54444734,"identity":"370b6795-65a3-408a-b72f-ac3f008ec730","added_by":"auto","created_at":"2024-04-10 16:12:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59600,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the confocal μ-XRF spectrometer.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/ec366e2a5ad1896a0fd571db.jpg"},{"id":54444726,"identity":"fe1d7120-ac1d-474b-a3a2-4b1d2f0bb12e","added_by":"auto","created_at":"2024-04-10 16:12:49","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45118,"visible":true,"origin":"","legend":"\u003cp\u003eOptical micrograph of a representative femur sample. The red square represents the scanning area.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/485ab765703e4bbb916d705a.jpeg"},{"id":54444733,"identity":"8842d070-ffeb-4e90-961b-6a544a410724","added_by":"auto","created_at":"2024-04-10 16:12:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57008,"visible":true,"origin":"","legend":"\u003cp\u003eA typical XRF spectrum of G2 femur measured by confocal μ-XRF.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/e683f3761683184967acdb93.jpg"},{"id":54444739,"identity":"f8fa416a-0fc9-4c68-abde-2f9485a736a6","added_by":"auto","created_at":"2024-04-10 16:12:53","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94709,"visible":true,"origin":"","legend":"\u003cp\u003eG1: (a) scanning region. Bio-distribution of (b) K; (c) Ca; (d) Fe; (e) Zn and (f) Sr.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/81de81fd3a95703a713c882d.jpeg"},{"id":54444730,"identity":"379d644f-e143-4b6a-a38c-a86ade33207a","added_by":"auto","created_at":"2024-04-10 16:12:50","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98646,"visible":true,"origin":"","legend":"\u003cp\u003eG2: (a) scanning region. (b) K; (c) Ca; (d) Fe; (e) Zn and (f) Sr.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/33a0d8efdf1cbd0093711326.jpeg"},{"id":54444732,"identity":"a85cece9-73d1-4b16-b97c-4bee4473b31c","added_by":"auto","created_at":"2024-04-10 16:12:51","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94039,"visible":true,"origin":"","legend":"\u003cp\u003eG3: (a) scanning region. (b) K; (c) Ca; (d) Fe; (e) Zn and (f) Sr.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/51e5756f158d8015aead15b4.jpeg"},{"id":54446239,"identity":"88c0a4eb-5e29-4e79-ab12-809391609fd8","added_by":"auto","created_at":"2024-04-10 16:20:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":516001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4180548/v1/e107c156-1d4f-4d59-b865-aed305c43437.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eElemental detection and mapping of rat bone matrix induced by chemoradiotherapy with confocal μ-XRF\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn 2020, female breast cancer emerged as the predominant cause of global cancer incidence, surpassing lung cancer, with an estimated 2.3\u0026nbsp;million new cases accounting for 11.7% of all reported cancer cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Common treatment strategies for breast cancer include chemotherapy and radiotherapy [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The administration of treatments such as chemotherapy and radiotherapy can induce temporary or non-temporary amenorrhea in premenopausal women, thereby reducing estrogen production. This hormonal alteration may contribute to accelerated bone loss, the development of osteoporosis, and an increased risk of fractures [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, it is imperative to assess the impact of chemotherapy and radiotherapy on bone loss and osteoporosis.\u003c/p\u003e \u003cp\u003eThe measurement of bone mineral density (BMD) is widely recognized as the gold standard in clinical practice for assessing the risk of osteoporotic fractures. In addition to potassium (K), calcium (Ca), and phosphorus (P), bones also contain various mineral microelements, including iron (Fe), zinc (Zn), strontium (Sr), among others. The presence of microelements is indispensable for the normal growth and development of the skeletal system in both humans and animals. Despite their minor role as building components in bones, microelements play crucial functional roles in bone metabolism and turnover, making them significant factors influencing the development of osteoporosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For instance, Fe accumulation, serving as an independent risk factor for osteoporosis, can significantly expedite bone loss in postmenopausal women afflicted with osteoporosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Tsay et al. conducted a Fe intervention in mice and found that the mice with accumulated Fe experienced severe bone loss and a significant decrease in bone density [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Jia et al. replicated the Fe intervention study and also discovered that excessive iron leads to bone loss, which is associated with the activation of osteoclasts and an increase in oxidative stress levels [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Zn can increase bone mass by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Sr can promote bone formation and inhibit bone resorption [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe traditional methods used for determining bone mineral microelements include flame atomic absorption spectrometry (FAAS), atomic fluorescence spectrometry (AFS), and inductively coupled plasma-based techniques (ICP) [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. FAAS, AFS, and ICP are destructive methods, because they require the samples to undergo chemical pretreatment (i.e., dissolution or digestion) before analysis. Compared with the elemental analysis method mentioned above, the X-ray fluorescence (XRF) method can simply, quickly, and nondestructively obtain content and distribution information of elements in samples. XRF has been extensively employed in the biomedical field to elucidate the intrinsic correlation between analysis outcomes and alterations in the physiological milieu of diseases, particularly for ascertaining the content and distribution of trace elements within bone [\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For instance, Gomez et al. employed synchrotron radiation X-ray fluorescence (SRXRF) to investigate the spatial distribution of zinc in human bone and pig osteogenesis, revealing a non-uniform distribution pattern with the highest concentration observed on the surface of haversian bone [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moise et al. conducted a longitudinal study using in vivo XRF to monitor the long-term intake of self-supplementing strontium citrate in osteoporotic women, and observed a continuous increase in Sr levels even after 4 years, highlighting the necessity of monitoring prolonged usage of Sr supplements [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Fei et al. employed SRXRF to investigate alterations in femur elemental composition in a diabetic osteoporosis animal model [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Their findings revealed a significant reduction in femur bone density among diabetic mice, accompanied by notable decreases in the relative mineral contents of Ca, P, Sr, and Zn; however, no significant differences were observed for Cr, Fe, Cu, and Pb.\u003c/p\u003e \u003cp\u003eThe biological samples often exhibit a multitude of components, intricate structures, and non-uniform distribution. XRF analysis of the entire sample solely reflects the average composition, while investigation of biochemical reactions occurring in the microenvironment necessitates analysis at the tissue or even cellular level. By quantifying the micro-distribution and content variations of elements within bone tissue and/or bone cells, a more comprehensive understanding of aberrant bone metabolism can be attained. The confocal \u0026micro;-XRF technique represents a significant advancement in XRF methodology, surpassing the conventional multielement analysis capabilities of traditional XRF methods. The confocal \u0026micro;-XRF technique offers notable advantages including exceptional sensitivity, precise detection, and micro-area analysis, enabling the acquisition of three-dimensional elemental distributions [\u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, our primary objective was to assess the impact of chemotherapy and radiotherapy on the femoral distribution of Ca, Fe, Zn, K, and Sr in rats using confocal \u0026micro;-XRF analysis. Additionally, we aimed to investigate the potential association between these determined microelements and bone loss.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eFifteen 12-week-old female Sprague Dawley (SD) rats, weighing 200 g each, were utilized for this study. The experimental animals used in this study were sourced from the Experimental Animal Center of Gannan Medical University. The rats were housed in ventilated cages maintained at standard environmental conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003eC, 50% \u0026plusmn; 5% humidity and a 12/12 h light/dark cycle) with ad libitum access to standard water and diet. The SD rats were randomly allocated into three groups (n\u0026thinsp;=\u0026thinsp;5 for each group), designated as G1, G2, and G3, respectively. The control group consisted of SD rats in G1 group. SD rats in the G2 group were administered a single intraperitoneal injection of docetaxel (15 mg/kg) and cyclophosphamide (60 mg/kg) chemotherapy drugs on the first day of each week for a duration of 4 weeks [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. On the 7th day of the final chemotherapy cycle (i.e., week 4), SD rats in the G2 group underwent thoracic radiotherapy that simulated breast cancer radiotherapy. The SD rats in the G3 group were not subjected to chemotherapy but instead received a single dose of 20 Gy radiation in the supine position using a linear accelerator Varian\u0026reg; (Clinac 2100). The X-ray beam had a nominal energy of 6 MV, and the dose rate was 240 cGy/min. The irradiation was administered to the left thoracic region of the rats within a 2 cm\u003csup\u003e2\u003c/sup\u003e field. The irradiation of SD rats with a dose of 20 Gy was found to be bioequivalent to the typically administered dose of 45 to 60 Gy in BC patients [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. After irradiation, SD rats were returned to their cages and subjected to daily monitoring for survival, dietary intake, and physical activity. SD rats in the G1 and G3 groups were administered a single intraperitoneal injection of 0.9% normal saline, with an equivalent volume as that given to the G2 group, on the first day of each week for four consecutive weeks. The experimental procedures involving animals in this study were conducted in strict accordance with the guidelines for the management and utilization of experimental animals published by the National Scientific and Technological Academic Works Publishing Fund Committee. Additionally, all protocols were approved by both the Ethics Committee and Animal Experiment Committee of Gannan Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sample preparation\u003c/h2\u003e \u003cp\u003eReferring to animal treatment in \u003cem\u003eRef.\u003c/em\u003e 29, the rats that had undergone fasting were humanely euthanized using pentobarbital anesthesia at the sixth week after the end of radiotherapy and chemotherapy. The right femurs in G1, G2, and G3 groups were promptly dissected and thoroughly cleared of any adherent soft tissue. The femurs were sectioned into 1 mm thick slices using a diamond blade and subsequently polished to achieve a flat surface. Confocal \u0026micro;-XRF analysis was conducted on the cancellous portion of the specimens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Confocal \u0026micro;-XRF\u003c/h2\u003e \u003cp\u003eThe elemental analysis of all femur samples was conducted using confocal \u0026micro;-XRF, and a schematic diagram illustrating the scanning spectrometer is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The confocal \u0026micro;-XRF spectrometer comprises an X-ray source, a polycapillary focusing X-ray lens (PFXRL), a polycapillary parallel X-ray lens (PPXRL), a sample stage, a charge-coupled device camera, an X-ray detector, a signal amplifier, a counting circuit, and a computer. The comprehensive elucidation of the confocal \u0026micro;-XRF technique can be found in our previously published work [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The detector is capable of detecting only XRF signals originating from the confocal microvolume formed by the overlapping of the PFXRL output focal spot and the PPXRL input focal spot. By precisely manipulating the sample stage, the confocal microvolume can be dynamically relocated within the specimen, facilitating high-resolution elemental scanning analysis in situ on a point-to-point basis. The samples in this experiment were scanned point-by-point using a dwell time of 20 s, a scanning area of 3\u0026times;3 mm\u003csup\u003e2\u003c/sup\u003e, and an effective step (pixel) size of 20 \u0026micro;m. The optical micrograph in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents a representative view of the femur sample, accompanied by a confocal \u0026micro;-XRF scanning area. The elemental concentration data from each pixel was collected and utilized to generate a corresponding distribution mapping of the elements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Elemental analysis\u003c/h2\u003e \u003cp\u003eThe excited XRF photons in the confocal microvolume are emitted isotropically. The relationship between the XRF intensity of element \u003cem\u003ei\u003c/em\u003e detected and its concentration can be expressed as follows [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${I}_{i}\\propto {S}_{i}\\bullet {C}_{i}\\bullet A$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e represents the net intensity of the characteristic XRF peak for element \u003cem\u003ei\u003c/em\u003e (counts per second, cps), \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e denotes the concentration of elemen \u003cem\u003ei\u003c/em\u003e (\u0026micro;g\u0026sdot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e signifies the sensitivity factor for analyzing element \u003cem\u003ei\u003c/em\u003e (cps\u0026sdot;cm\u0026sdot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cem\u003eA\u003c/em\u003e represents the absorption factor for element \u003cem\u003ei\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe confocal \u0026micro;-XRF system was calibrated using a standard sample of NIST (National Institute of Standards and Technology, USA) bone meal to obtain the above experimental parameters. The inorganic components of bone are mainly Ca and P, and trace elements are K, Fe, Zn, Sr, etc. For the bone meal NIST SRM-1486, the certified Ca concentration is 26.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24%. In this study, about 100 mg of bone meal NIST SRM-1486 was pressed to obtain a tablet with a thickness of 1 mm.\u003c/p\u003e \u003cp\u003eThe sensitivity and minimum detection limits (MDL) for each element were determined by analyzing standard bone meal samples purchased from Shenzhen Deborui Biological Technology Co., LTD. The standard samples were placed on 6 \u0026micro;m thick Mylar membranes. The blank spectrum of the Mylar film was processed, and subsequently, the calculated concentrations of elements in the samples were adjusted by subtracting the corresponding blank values.\u003c/p\u003e \u003cp\u003eThe X-ray spectra were subjected to peak fitting using the AXIL program package in order to extract the net intensities of XRF peaks [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. One of five femur samples in each group were randomly selected and were scanned by Confocal \u0026micro;-XRF microprobe for element analysis. The data were removed if they were less than three times the corresponding standard deviation. The elemental peak area was normalized to the peak counts of Ar, which is present in air at a constant proportion. The normalized peak areas were utilized to estimate the relative elemental contents. The sample thickness, measurement geometry, and beam energy remained unchanged to ensure the consistent application of element-specific conversion factors across all measurements.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eThe MDL for element \u003cem\u003ei\u003c/em\u003e can be expressed as [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]:\u003c/p\u003e\n\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ2\" class=\"mathdisplay\"\u003e$${m}_{MDL}=3\\bullet {C}_{i}\\bullet \\frac{\\sqrt{{I}_{B}}}{{I}_{i}}$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eHere, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e represents the net fluorescent line intensity and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e denotes the background intensity of the considered element \u003cem\u003ei\u003c/em\u003e. The MDL of the confocal \u0026micro;-XRF spectrometer for the target elements can be found in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eMinimum detection limit (MDL) for target elements (\u0026micro;g/g).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eElements\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({m}_{MDL}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCa\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e149\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eK\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e14.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFe\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZn\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSr\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e22.931\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows a typical XRF spectrum of G2 femur measured by confocal \u0026micro;-XRF method, which encompasses characteristic X-rays emitted by various elements present in the bone matrix. The XRF spectrum revealed the presence of several microelements, including S, K, Mn, Fe, Cu, Zn, Pb and Sr in the femur sample. This finding suggests that confocal \u0026micro;-XRF can effectively and simultaneously detect a wide range of trace elements in the analyzed samples. The spatial distribution of K, Ca, Fe, Zn, and Sr within the scanning region obtained through confocal \u0026micro;-XRF analysis is depicted in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e to \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. The visual analysis of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026thinsp;~\u0026thinsp;6 reveals that the distribution of Ca was relatively homogeneous, while the distributions of K, Fe, Zn, and Sr exhibited non-uniform patterns. Notably, a significant deposition of iron in bone matrix was observed specifically in the G2 group. The two-dimensional distribution of elements matches the morphological characteristics, which can reveal the elemental correlations of different femurs. The average relative element contents of the investigated elements in the scanning region of G1, G2 and G3 are given in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Compared to the G1 group, significant calcium loss was observed in both the G2 and G3 groups. The average relative element contents reveal no significant differences in K, Zn, and Sr; however, notable disparities are observed in Fe. This suggests that chemotherapy induces overexpression of Fe, leading to competition with calcium ions. Fe serves as a cofactor in enzymes involved in the synthesis of collagen bone matrix, as well as in 25 OH vitamin D hydroxylase, an enzyme responsible for activating vitamin D and facilitating calcium absorption [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe objective of this study was to examine the impact of chemotherapy and radiotherapy on the elemental composition and distribution within the bone matrix. Relevant literature has demonstrated that radiotherapy and chemotherapy have the potential to induce alterations in bone mineral density [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. For instance, Nogueira et al. conducted a micro-XRF analysis to assess the impact of photon irradiation treatment on calcium levels in the dorsal ribs of Wistar rats and observed a significant decrease in calcium content following radiotherapy [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our radiotherapy findings were consistent with the results obtained by Nogueira et al. However, to the best of our knowledge, there is currently no assessment available regarding crucial microelements in the bone matrix after chemotherapy. The results of our study demonstrate the efficacy of confocal \u0026micro;-XRF method in analyzing crucial major and trace elements, including Ca, Fe, Zn, K, and Sr within the bone matrix.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eRelative element contents (\u0026micro;g/g) in femur of control and chemotherapy/radiotherapy rat.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCa\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFe\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eZn\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSr\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e33.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e251.13\u0026thinsp;\u0026plusmn;\u0026thinsp;11.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e114.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e81.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e37.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e113.20\u0026thinsp;\u0026plusmn;\u0026thinsp;11.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e25.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e96.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e100.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e25.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e121.31\u0026thinsp;\u0026plusmn;\u0026thinsp;11.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e13.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e107.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e91.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePostmenopausal osteoporosis can be considered as a biochemical phenomenon characterized by the presence of coarse and shortened bone mass, along with weakened microstructure resulting from the loss of estrogen\u0026rsquo;s direct impact on osteoclasts [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, the biochemical association between breast cancer and postmenopausal osteoporosis is a matter of significant concern and scientific interest. Both bone and breast tissue rely on estrogen, a pivotal hormone that regulates bone density and maintains a delicate equilibrium between bone formation and resorption by modulating osteoclast levels or promoting osteoblast proliferation [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. Chemotherapy and radiotherapy-induced ovarian failure results in a decline in estrogen levels, subsequently leading to significant calcium loss and an elevation in bone iron content.\u003c/p\u003e\n\u003cp\u003eThe enhanced survival rates of breast cancer patients undergoing chemotherapy and radiotherapy underscore the significance of comprehending the mechanisms and long-term impacts of chemotherapy and radiotherapy-induced alterations in bone composition, encompassing both major and trace elements. The specific mechanism of Fe accumulation leading to postmenopausal osteoporosis remains unclear. The cellular-level studies have demonstrated that the accumulation of Fe can impede the activity and functionality of osteoblasts, stimulate osteoclast function, induce apoptosis in mesenchymal stem cells, and hinder their differentiation into osteoblasts [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIt should be noted that the spatial resolution achieved with the confocal \u0026micro;-XRF method is limited, which precludes studying structures at the (sub-) cellular level. In future investigations, our aim is to employ nano-XRF techniques for the detection of Fe distribution within osteoblasts and elucidate the underlying cellular and/or molecular mechanisms. This will provide profound insights into the impact of Fe metabolism on osteoporosis following breast cancer chemotherapy.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe present study demonstrates the application of confocal \u0026micro;-XRF as an analytical method for assessing the impact of chemotherapy and radiotherapy on alterations in content and distribution patterns of essential major and trace elements within bone matrix. The results suggest a significant decrease in Ca levels following chemotherapy and radiotherapy, indicating the presence of osteoporosis. In addition, chemotherapy results in a significant accumulation of iron in bone matrix.\u003c/p\u003e \u003cp\u003eIn conclusion, confocal \u0026micro;-XRF has emerged as a robust analytical method for assessing the impact of chemotherapy and radiotherapy on osteoporosis. Chemotherapeutic drugs such as cyclophosphamide and docetaxel induce ovarian changes that result in reduced or insufficient estrogen levels, subsequently leading to alterations in bone matrix composition and the promotion of osteoporosis. The primary constraint of the proposed confocal \u0026micro;-XRF method lies in its current limitation to preclinical testing exclusively in rats, rendering it ethically unfeasible for human experimentation and thereby diminishing its clinical applicability for population-wide screening and treatment.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQiuxia Li\u003c/strong\u003e: Conceptualization, Methodology, Validation, Investigation, Formal analysis, Data curation, Writing-original draft. \u003cstrong\u003eHongchi Chen\u003c/strong\u003e: Writing-review \u0026amp; editing. \u003cstrong\u003eLazhen Zhou\u003c/strong\u003e: Writing-review \u0026amp; editing. \u003cstrong\u003eFangzuo Li\u003c/strong\u003e: Conceptualization, Writing-review \u0026amp; editing, Supervision, Funding acquisition, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science Foundation of Jiangxi Province (Grant No. 20224BAB201020), and the Startup Fund of High-level Talents Scientific Research of Gannan Medical University (QD201805).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209-249\u003c/li\u003e\n\u003cli\u003eKim DY, Youn JC, Park MS et al (2019) Cardiovascular outcome of breast cancer patients with concomitant radiotherapy and chemotherapy: A 10-year multicenter cohort study. J Cardiol 74:175-181\u003c/li\u003e\n\u003cli\u003eKozak MM, Eyben RV, Gutkin PM et al (2021) Locoregional and Distant Outcomes in Women With cT1-3N1 Breast Cancer Treated With Neoadjuvant Chemotherapy With or Without Adjuvant Radiotherapy. Clin Breast Cancer 21:373-382\u003c/li\u003e\n\u003cli\u003eDi L, Liu LJ, Yan YM et al (2019) Discovery of a natural small molecule compound that suppresses tumor EMT, stemness and metastasis by inhibiting TGF\u0026beta;/BMP signaling in triple-negative breast cancer. J Exp Clin Cancer Res 38:134\u003c/li\u003e\n\u003cli\u003eZhang L, Yang F, Huang L et al (2017) Luteolin enhances the antitumor activity of lapatinib in human breast cancer cells. Biomed Res 28:4902-4907\u003c/li\u003e\n\u003cli\u003eStumpf U, Kostev K, Siebenb\u0026uuml;rger G et al (2020) Influence of chemotherapy and endocrine treatment on fractures in postmenopausal women with breast cancer-a retrospective cohort study. J Bone Oncol 22:100292\u003c/li\u003e\n\u003cli\u003eVestergaard P (2019) Drugs Causing Bone Loss, Handbook of experimental pharmacology.\u003c/li\u003e\n\u003cli\u003eZofkova I, Nemcikova P, Matucha P (2013) Trace elements and bone health. Clin Chem Lab Med 51:1555-1561\u003c/li\u003e\n\u003cli\u003eZheng J, Mao X, Ling J et al (2014) Low serum levels of zinc, copper, and iron as risk factors for osteoporosis: a meta-analysis. Biol Trace Elem Res 160:15-23 \u003c/li\u003e\n\u003cli\u003eWeinberg ED (2006) Iron loading: a risk factor for osteoporosis. Biometals 19:633-635\u003c/li\u003e\n\u003cli\u003eTsay J, Yang Z, Ross FP et al (2010) Bone loss caused by iron overload in a murine model: importance of oxidative stress. Blood 116:2582-2589\u003c/li\u003e\n\u003cli\u003eJia P, Xu YJ, Zhang ZL et al (2012) Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species. J Orthop Res 30:1843-1852 \u003c/li\u003e\n\u003cli\u003eYamaguchi M, Goto M, Uchiyama S et al (2008) Effect of zinc on gene expression in osteoblastic MC3T3-E1 cells: enhancement of Runx2, OPG, and regucalcin mRNA expressions. Mol Cell Biochem 312:157-166\u003c/li\u003e\n\u003cli\u003eHie M, Iitsuka N, Otsuka T et al (2011) Zinc deficiency decreases osteoblasts and osteoclasts associated with the reduced expression of Runx2 and RANK. Bone 49:1152-1159\u003c/li\u003e\n\u003cli\u003eYamaguchi M, Segawa Y, Shimokawa N et al (1992) Inhibitory effect of beta-alanyl-L-histidinato zinc on bone resorption in tissue culture. Pharmacology 45:292-300\u003c/li\u003e\n\u003cli\u003eBonnelye E, Chabadel A, Saltel F et al (2008) Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. Bone 42:129-138\u003c/li\u003e\n\u003cli\u003eMarie PJ, Felsenberg D, Brandi ML (2011) How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis. Osteoporosis Int 22:1659-1667\u003c/li\u003e\n\u003cli\u003eMarx D, Yazdi AR, Papini M et al (2020) A review of the latest insights into the mechanism of action of strontium in bone. Bone Rep 12:100273\u003c/li\u003e\n\u003cli\u003eLouis O, Winke PVD, Covens P et al (1994) Mineral content of vertebral trabecular bone: Accuracy of dual energy quantitative computed tomography evaluated against neutron activation analysis and flame atomic absorption spectrometry. Bone 15:35-39\u003c/li\u003e\n\u003cli\u003e\u0026Scaron;čančar J, Milačič R, Benedik M et al (2000) Determination of trace elements and calcium in bone of the human iliac crest by atomic absorption spectrometry. Clin Chim Acta 293:187-197\u003c/li\u003e\n\u003cli\u003eMahanti HS, Barnes RM (1983) Determination of major, minor and trace elements in bone by inductively-coupled plasma emission spectrometry. Anal Chim Acta 151:409-417\u003c/li\u003e\n\u003cli\u003eGomez S, Rizzo R, Mucelli MP et al (1999) Zinc Mapping in Bone Tissues by Histochemistry and Synchrotron Radiation-Induced X-Ray Emission: Correlation With the Distribution of Alkaline Phosphatase. Bone 25:33-38\u003c/li\u003e\n\u003cli\u003eMoise H, Adachi JD, Chettle DR et al (2012) Monitoring bone strontium levels of an osteoporotic subject due to self-administration of strontium citrate with a novel diagnostic tool, in vivo XRF: A case study. Bone 51:93-97\u003c/li\u003e\n\u003cli\u003eMoise H, Chettle DR, Milić AP (2014) Monitoring bone strontium intake in osteoporotic females self-supplementing with strontium citrate with a novel in-vivo X-ray fluorescence based diagnostic tool. Bone 61:48-54\u003c/li\u003e\n\u003cli\u003eWohl GR, Chettle DR, Milić AP et al (2013) Accumulation of bone strontium measured by in vivo XRF in rats supplemented with strontium citrate and strontium ranelate. Bone 52:63-69\u003c/li\u003e\n\u003cli\u003eSales E, Silva CERD, Letichevsky S et al (2020) Study of composition and structure of demineralized bone using X-ray techniques. Radiat Phys Chem 167:108310\u003c/li\u003e\n\u003cli\u003eZhang XX, Specht AJ, Wells E et al (2021) Evaluation of a portable XRF device for in vivo quantification of lead in bone among a US population. Sci Total Environ 753:142351\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez HJ, Valentinuzzi MC, Gren\u0026oacute;n M et al (2008) Total reflection X-ray fluorescence analysis of oral fluids of women affected by osteoporosis and osteopenia. Spectrochim Acta B 63:1485-1488\u003c/li\u003e\n\u003cli\u003eFei YR, Zhang M, Li M et al (2007) Element analysis in femur of diabetic osteoporosis model by SRXRF microprobe. Micron 38:637-642\u003c/li\u003e\n\u003cli\u003eBallarre J, Desimone PM, Chorro M et al (2013) Bone quality around bioactive silica-based coated stainless steel implants: Analysis by Micro-Raman, XRF and XAS techniques. J Struct Biol 184:164-172\u003c/li\u003e\n\u003cli\u003eHahn M, Buzanich G, J\u0026auml;hn K et al (2019) Analysis of cobalt deposition in periprosthetic bone specimens by high-resolution synchroton XRF in undecalcified histological thin sections. Materialia 6:100290\u003c/li\u003e\n\u003cli\u003eMazel V, Reiche I, Busignies V et al (2011) Confocal micro-X-ray fluorescence analysis as a new tool for the non-destructive study of the elemental distributions in pharmaceutical tablets. Talanta 85:556-561\u003c/li\u003e\n\u003cli\u003eNakazawa T, Tsuji K (2013) Depth-selective elemental imaging of micro SD card by confocal micro XRF analysis. X-Ray Spectro 42:123-127\u003c/li\u003e\n\u003cli\u003eL\u0026uuml;hl L, Mantouvalou I, Schaumann I et al (2013) Three-dimensional chemical mapping with a confocal XRF setup. Anal Chem 85:3682-3689\u003c/li\u003e\n\u003cli\u003eNakano K, Nishi C, Otsuki K et al (2011) Depth elemental imaging of forensic samples by confocal micro-XRF method. Anal Chem 83:3477-3483\u003c/li\u003e\n\u003cli\u003eYi L, Qin M, Wang K et al (2016) The three-dimensional elemental distribution based on the surface topography by confocal 3D-XRF analysis. Appl Phys A 122:856-862\u003c/li\u003e\n\u003cli\u003eXu Q, Xia W, Zhou L et al (2022) Determination of hepatic iron deposition in drug-induced liver fibrosis in rats by confocal micro-XRF spectrometry. ACS Omega 7:3738-3745\u003c/li\u003e\n\u003cli\u003eKonat GW, Kraszpulski M, James I et al (2008) Cognitive Dysfunction Induced by Chronic Administration of Common Cancer Chemotherapeu-tics in Rats. Metab Brain Dis 23:325-333\u003c/li\u003e\n\u003cli\u003eKr\u0026uuml;se JJ, Zurcher C, Strootman EG et al (2011) Structural changes in the auricles of the rat heart after local ionizing irradiation. Radiother Oncol 58:303-311\u003c/li\u003e\n\u003cli\u003eBertin EP (1975) Principles and Practice of X-ray Spectrometric Analysis, Secondy. Plenun Press, London\u003c/li\u003e\n\u003cli\u003eBernasconi G, Tajani A, Kregsamer P (2000) Manual for QXAS/AXIL. Vers\u0026atilde;o 3.5. International Atomic Energy Agency, Vienna\u003c/li\u003e\n\u003cli\u003eSamber BD, Silversmit G, Schamphelaere KD et al (2010) Element-to-tissue correlation in biological samples determined by three-dimensional x-ray imaging methods. J Anal At Spectrom 25:544-553\u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Amelio P, Cristofaro MA, Tamone C et al (2008) Role of iron metabolism and oxidative damage in postmenopausal bone loss. Bone 43:1010-1015\u003c/li\u003e\n\u003cli\u003eSato K, Nohtomi K, Demura H et al (1997) Saccharated ferric oxide (SFO)-induced osteomalacia: in vitro inhibition by SFO of bone formation and 1,25-dihydroxy-vitamin D production in renal tubules. Bone 21:57-64\u003c/li\u003e\n\u003cli\u003eNogueira LP, Barroso RC, Almeida APD et al (2012) Evaluation of Photon Irradiation Treatment Upon Calcium Content of Ribs of Wistar Rats Using Micro-XRF. AIP Conference Proceedings 1437:104-107.\u003c/li\u003e\n\u003cli\u003eRusz O, Kah\u0026aacute;n Z (2013) Bone Homeostasis and Breast Cancer: Implications for Complex Therapy and the Maintenance of Bone Integrity. Pathol Oncol Res 19:1-10\u003c/li\u003e\n\u003cli\u003eMuhammad A, Mada SB, Malami I et al (2018) Postmenopausal osteoporosis and breast cancer: The biochemical links and beneficial effects of functional foods. Biomed Pharmacother 107:571-582.\u003c/li\u003e\n\u003cli\u003eTr\u0026eacute;mollieres FA (2014) Screening for osteoporosis after breast cancer: For whom, why and when. Maturitas 79:343-348\u003c/li\u003e\n\u003cli\u003eCauley JA (2015) Estrogen and bone health in men and women. Steroids 99:11-15\u003c/li\u003e\n\u003cli\u003eManolagas SC (2010) From Estrogen-Centric to Aging and Oxidative Stress: A Revised Perspective of the Pathogenesis of Osteoporosis. Endocr Rev 31:266-300\u003c/li\u003e\n\u003cli\u003eKhalid AB, Krum SA (2016) Estrogen receptors alpha and beta in bone. Bone 87:130-135\u003c/li\u003e\n\u003cli\u003eEastell R, Szulc P (2017) Use of bone turnover markers in postmenopausal osteoporosis. Lancet Diabetes Endo 5:908-923\u003c/li\u003e\n\u003cli\u003eYang Q, Jian JL, Abramson SB, et al (2011) Inhibitory effects of iron on bone morphogenetic protein 2-induced osteoblastogenesis. J Bone Miner Res 26:1188-1196\u003c/li\u003e\n\u003cli\u003eBalogh E, Tolnai E, B\u0026eacute;laNagy J, et al (2016) Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin. BBA-Mol Basis Dis 1862:1640-1649\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Elemental analysis/imaging, Confocal µ-XRF, Bone matrix, Chemotherapy, Radiotherapy","lastPublishedDoi":"10.21203/rs.3.rs-4180548/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4180548/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer is among the most prevalent malignant tumors in women. The administration of chemotherapy and radiotherapy for breast cancer treatment can lead to amenorrhea and substantial bone loss in women. Consequently, postmenopausal women are susceptible to the morbidity and mortality risks associated with breast cancer and postmenopausal osteoporosis. In this study, we investigated the impact of chemotherapy and radiotherapy on osteoporosis in female rats using confocal microbeam X-ray fluorescence (\u0026micro;-XRF) method. The female Sprague Dawley (SD) rats were categorized into three groups: the control group (G1), the chemotherapy and radiotherapy group (G2), and the radiotherapy-only group (G3). The SD rats were euthanized six weeks post chemotherapy and radiotherapy, and femur slices with a thickness of 1mm were obtained for confocal \u0026micro;-XRF analysis. The results demonstrate a significant loss of calcium in the G2 and G3 groups, while the G2 group exhibited a substantial increase in Fe content compared to the G1 group. The conclusion can be drawn that the occurrence of osteoporosis is related to chemotherapy and radiotherapy, while the significant elevation in bone iron content signifies the progression of osteoporosis.\u003c/p\u003e","manuscriptTitle":"Elemental detection and mapping of rat bone matrix induced by chemoradiotherapy with confocal μ-XRF","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 16:12:39","doi":"10.21203/rs.3.rs-4180548/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2024-05-21T10:21:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-05T06:48:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-29T07:27:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Medical Research","date":"2024-03-28T08:05:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1780aedd-7fc4-4e6a-90ff-9f78802ba5ff","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-10T16:12:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 16:12:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4180548","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4180548","identity":"rs-4180548","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}