Colorectal cancer and trace elements alteration

Journal Pre-proof COLORECTAL CANCER AND TRACE ELEMENTS ALTERATION ´ Jovan T. Juloski, Aleksandar Rakic, Vladica V. Cuk, Vladimir M. – ´ Cuk, Srdan Stefanovi´c, Dragica Nikoli´c, Saˇsa Jankovi´c, Alexander M. Trbovich, Silvio R. De Luka

PII:

S0946-672X(19)30377-3

DOI:

https://doi.org/10.1016/j.jtemb.2020.126451

Reference:

JTEMB 126451

To appear in:

Journal of Trace Elements in Medicine and Biology

Received Date:

5 June 2019

Revised Date:

11 December 2019

Accepted Date:

5 January 2020

´ ´ Please cite this article as: Juloski JT, Rakic A, Cuk VV, Cuk VM, Stefanovi´c S, Nikoli´c D, Jankovi´c S, Trbovich AM, De Luka SR, COLORECTAL CANCER AND TRACE ELEMENTS ALTERATION, Journal of Trace Elements in Medicine and Biology (2020), doi: https://doi.org/10.1016/j.jtemb.2020.126451

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COLORECTAL CANCER AND TRACE ELEMENTS ALTERATION

Jovan T. Juloski, MDa, Aleksandar Rakic, MDb, Vladica V. Ćuk, MDa, Vladimir M. Ćuk, MD, PhDa, Srđan Stefanović, PhDc, Dragica Nikolić, PhDc, Saša Janković, PhDc, Alexander M. Trbovich, MD, PhDb, Silvio R. De Luka, MD, PhDb* Zvezdara Medical University Center, Surgery Clinic “Nikola Spasić”, Dimitrija Tucovica 161, 11000 Belgrade, Serbia b Department of Pathological Physiology, Faculty of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia c Institute of Meat Hygiene and Technology, Kaćanskog 13, 11000 Belgrade, Serbia

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E-mail adresses of authors:

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*corresponding author Silvio R. De Luka [email protected] Department of Pathological Physiology, Faculty of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia Tel. +381112685340

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Jovan T. Juloski - [email protected] Aleksandar Rakić - [email protected] Vladica V. Ćuk - [email protected] Vladimir M. Ćuk - [email protected] Srđan Sretenović - [email protected] Dragica Nikolić - [email protected] Saša Janković - [email protected] Alexander M. Trbovich - [email protected]

Highlights

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Significant alterations of Na, K, Mg, Ca, Cu, Zn, Se, Mn, Cd, Cr and Hg in malignant tissue of colorectal cancer (CRC) compared to adjacent healthy bowel tissue were found Cu/Zn ratio was significantly higher in CRC tissue compared to adjacent healthy

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intestinal tissue and patients with higher CRC stages had significantly higher Cu/Zn

Male subjects with CRC had significantly higher Ca2+ tissue levels compared to

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ratio

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female subjects with CRC

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Abstract

Background: Trace elements have important influence on body function primarily because of the vital role they have in many physiological processes. Their

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alterations have been found in many disorders, including cancer. It has been well known for decades that disturbances in elemental concentration may lead to cell damaging, DNA injuries and imbalance in oxidative burden.Our study tried to determine the difference of trace elements concentrations between colorectal adenocarcinoma and adjacent healthy intestinal tissue.

Methods: 59 subjects participated in this study. Healthy colon mucosa samples and colon tumor tissue samples were obtained from patients previously diagnosed with colon carcinoma by standard diagnostic procedures. Analysis of the elements was performed by inductively coupled plasma mass spectrometry (ICP-MS). Results: The results showed that Na, K, Mg, Ca, Cu, Zn, Se, Mn, Cd, Cr and Hg

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significantly differ between malignant tissue of colorectal cancer and adjacent

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healthy bowel tissue. We have, also, found that Cu/Zn tissue ratio was significantly

stages had also significantly higher ratio.

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higher in CRC compared to a healthy tissue and that patients with higher CRC

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Conclusions: Since this is the first such study in Balkan region, we assume that

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results of our study could be a good indicator of elemental alterations in colorectal cancer of Balkan population, due to similarity in lifestyle, dietary intake, pollution

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and exposure to toxic elements.

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Keywords: colorectal cancer, trace elements, healthy tissue, Cu/Zn ratio

Introduction In optimal concentration ranges, chemical elements are indispensable for vital functions of human body on cellular and molecular levels. Chemical elements, also called „building blocks“, are essential participants in the processes of development, metabolism and adaptation to ever changing environmental conditions[1,2].

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There are many different classifications of chemicals found in human body.

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One of them divides elements based on the percentage of their concentration in

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human body into: macroelements (concentration in body exceeds 0.01%), trace elements (concentration ranges from 0.00001% to 0.01%) and ultratrace elements,

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which concentration is lower than 0.00001%[2]. Maybe even more important is the

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classification of elements based on their physiological role in human organism. Structural elements are macroelements that are composing bulk of cell walls and

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membranes. Also, based on their physiological role, a group of trace elements is called essential. Element is considered essential to an organism when reduction of its exposure below certain limit results consistently in a reduction in a

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physiologically important function or when the element is integral part of an organic structure, performing a vital function in the organism because of the immense influence they have on many body functions[3,4]. That being said, even with very low concentrations, essential trace elements have a tremendous influence

on body function primary because of the integral part they have in many enzymatic systems. All around the world, cancer incidence and mortality are rapidly growing. Over 1.8 milion new cases of colorectal cancer (CRC) and 881000 deaths were estimated to occur in 2018 worldwide [5]. In the Republic of Serbia colorectal

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cancer is the second most frequent type of cancer in both males and females with

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about 4000 new cases and 2500 deaths annually [6].

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Elemental alterations have been found in many human diseases, including cancer. It has been well known for decades that disturbances in elemental

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concentration may lead to cell damaging, DNA injuries and imbalance in oxidative

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burden [7–9]with both of the latter mentioned processes can cause malignant transformation[10]. In the last few years there has been an emerging interest for

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understanding the exact role of trace elements and metals in pathogenesis of many human cancers, including colorectal cancer. Few studies were investigating elemental fingerprint in intestinal cancerous tissue and compared it to adjacent

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non-tumorous tissues as well with healthy controls with inconsistent results[7,11– 13].

Therefore, the aim of this study was to investigate the difference of various

macro- and trace elements concentrations between colorectal malignant tissue and adjacent healthy intestinal tissue. To our knowledge this is the first such study in

this region. In order to explain the eventual variation in levels of trace elements between tumorous and healthy tissue in colorectal region, we have included patient’s age, sex, and different stages of CRC as a potential causes of mentioned variations.

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Materials and methods

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Patients (n=59) with confirmed diagnosis of colorectal carcinoma participated

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in this study. Human cancer and healthy tissues were collected in accordance with the Ethics committee approval (Office for Human Research Protections, University

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Medical Center “Zvezdara”, Belgrade, Serbia). Prior to their enrolment in this

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study, all participants signed the informed consent. Healthy colon mucosa samples and colon tumor tissue samples were obtained from patients previously diagnosed

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with colon carcinoma by standard diagnostic procedures. All patients went through a standard preoperative colon preparation with oral medication and enemas in order for the colon to be free of a residual content. Approximately 1 g of both

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healthy colon tissue and tumor tissue were cut and placed in 2 ml micro-centrifuge sterile tubes. After surgical resection, the specimens were opened, abundantly rinsed with sterile saline solution and placed in sterile tubes. Tubes with collected tissues were frozen in liquid nitrogen and stored at –80 C until further use.

Sample preparation and reagents Frozen samples were thawed at +4 ºC for a day before analysis and then homogenized. An amount, approximately 0.5 g, of each thawed, homogenized tissue, was transferred into a teflon vessel with 5 mL nitric acid (67% Trace Metal Grade, Fisher Scientific, Bishop, UK) and 1.5 mL hydrogen peroxide (30%

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analytical grade, Sigma-Aldrich, St. Louis, MA, USA) for microwave digestion.

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The microwave (Start D, Milestone, Sorisole, Italy) program consisted of three

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steps: 5 min from room temperature to 180°C, 10 min hold at 180°C, 20 min vent. After cooling, the digested sample solutions were quantitatively transferred into

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disposable flasks and diluted to 100 mL with deionized water produced by a water

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purification system (Purelab DV35, ELGA, Buckinghamshire, UK). Analysis of the following nineteen elements: Fe, Zn, Cu, Mn, Se, Cr, Co, Ni,

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Na, K, Mg, Ca, Cd, Pb, Hg, As, U, Sn and Al was performed by inductively coupled plasma mass spectrometry (ICP-MS), (iCap Q mass spectrometer, Thermo Scientific, Bremen, Germany). The most abundant isotopes were used for

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quantification.

Torch position, ion optics and detector settings were re-adjusted daily using

tuning solution (Tune B, Thermo Scientific), in order to optimize mechanical and electrical parameters and minimize possible interference. Basic operating conditions of the instrument were: RF power (1550 W); cooling gas flow (14

L/min); nebulizer flow (1 L/min); collision gas flow (1 mL/min); operating mode (Kinetic Energy Discrimination-KED); dwell time (10 ms). Standard stock solutions containing 1000 mg/L of each element (Fe, Zn, Cu, Mn, Se, Cr, Co, Ni, Na, K, Mg, Ca, Cd, Pb, Hg and As) were purchased from Reagecon (Shannon, Co. Clare, Ireland). These solutions were used to prepare

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standards for five-point calibration curves (including zero). Multielement internal

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standard (6Li, 45Sc, 71Ga, 89Y, 209Bi) was introduced online by an additional line

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through the peristaltic pump, and covered a wide mass range. All solutions (standards, internal standards and samples) were prepared in 2% nitric acid.

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Quality assurance

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The quality of the analytical process was verified by analysis of the certified reference material NIST 1577c (bovine liver, Gaithersburg, MD, USA). Reference

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material was prepared as samples using microwave digestion. Measured concentrations were corrected for response factors of internal standards using the interpolation method and were within the range of the certified values for all

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isotopes (Table 1). No information was given regarding Hg, Al, Sn and U in the reference material and, therefore, analytical recoveries of 97-105% were determined using spiked samples. Statistical analysis

Data was analyzed using EZR for Mac OS X by R Studioand SPSS software version 20 (IBM Corp. Released 2011. IBM SPSS Statistics for Macintosh, Version 22.0. Armonk, NY: IBM Corp.).The characteristics of the subjects were analyzed using descriptive statistics (frequencies, ranges, means and standard deviations, SDs). Kolomogorov – Smirnov test was used to determine the

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normality of the data distribution. The paired t test and One-Way ANOVA were

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used for comparison of normally distributed variables, while the Wilcoxon Signed

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Ranks Test and Mann – Whitney Test were used for variables without normal distribution. Spearman’s Rank Correlation Test was used for investigation of

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was considered to be p  0.05.

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correlations between elements. In all analyses, the significant level of difference

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Results

Of 59 samples, 29 (49.2%) belonged to males, while 30 (50.8%) belonged to female patients. Mean patient age was 6710 years. Subjects were classified in two

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age groups: middle-aged ( 65 years) and older subjects (> 65 years). According to age and sex, our patients were assembled in four categories: older men, middleaged men, older women and middle-aged women. Adenocarcinoma was the histological type of CRC in all patients. The majority of patients were CRC stage III. The characteristics of the subjects in our study are presented in Table 2.

Comparison between concentrations of elements in tumor affected tissue and adjacent healthy tissue is presented in Table 3. Zn was significantly lower in malignant tissue while Cu, Cd, Mg, Se and Ca showed significantly higher concentrations in tumorous compared to a healthy tissue. Potassium was significantly higher in malignant tissue while sodium was significantly lower in

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malignant tissue compared to a healthy adjacent tissue. Chromium and mercury

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Ni and Sn showed no difference between two tissues.

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(Hg) were significantly higher in CRC, while levels of Mn, Fe, Al, Pb, U, As, Co,

Differences in elemental levels between CRC and adjacent healthy tissue in

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male and female subjects are presented in Tables 4 and 5, respectively. Elemental

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concentrations in malignant tissue showed no significant difference between sexes. Differences in elemental levels between CRC and adjacent healthy tissue in middle

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– aged and elderly subjects are presented in Tables 6 and 7. Also, there was no significant difference in concentrations of elements in malignant tissue between elderly patients and middle-aged adults.

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Cu/Zn ratio was significantly higher in CRC compared to a healthy adjacent tissue (p <0.001) (Table 8). In males, elderly and middle-aged subjects this trend of significance continued. On the other hand, in females and when compared between sexes and between age groups, the difference in Cu/Zn ratio showed no statistical significance (Table 8). Moreover, Cu/Zn ratio was significantly higher in patients

with CRC stages III and IV compared to those in lower stages (stages I and II) (Figure 1).In all subjects, the Cu/Zn ratio differed between stages of CRC but not significantly (p = 0.05, Figure 2). Interestingly, in female subjects, there was a significant difference in the Cu/Zn ratio when compared between stages of CRC, with the highest values of this ratio being in Stage IV (Figure 3).

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There was no significant difference in concentrations of any element measured

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in malignant tissue between any of age-sex formed categories of subjects (results

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not presented). Correlations of elements in CRC and elements in healthy tissue, as well as between CRC and healthy tissue are shown in Tables 9 and 10,

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respectively. In all of the subjects, none of the elements correlated with the

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Discussion

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subject's age.

Alterations of essential TE in CRC

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Cancerous tissue showed lower Zn level compared to healthy intestinal tissue in our study. This was also the case with male subjects, while in females there was no significant difference in Zn levels between the two types of tissue. Zn is an important component of more than 300 enzymes, many of them required for processes vital for synthesis of DNA and RNA, and an essential element for proper

immune function [14]. Zn, along with Mn and Cu, is a crucial part of the first line antioxidant defense enzymes - superoxide-dismutases (SODs)[15,16]. One study found significantly lower activity of antioxidant enzymes, along with significantly higher activity of pro-oxidant enzymes in CRC compared to healthy controls [16]. On the other hand, in excessive concentrations, Zn and Cu show pro-oxidant

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characteristics by generating reactive oxygen species (ROS) and, in that way,

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inducing cell damage [17,18]. It was shown that Zn could cause a growth arrest in

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colorectal cancer cells by disrupting cellular microtubule stability and by stabilizing the levels of the wildtype adenomatous polyposis coli (APC)

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protein [19]. There is inconsistency in differences of Zn levels between malignant

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and healthy intestinal tissue in similar studies, with some of them showing no significant difference[12,15] while a recent one showed lower zinc levels in

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malignant tissue[7]. It was proposed that lower levels of Zn in liver metastasis compared to healthy tissue were due to increased utilization of Zn by cancerous tissue and changes in Zn transporters [20,21] and it could explain lower Zn

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concentrations in malignant tissue in our study. Furthermore, as Zn has essential roles in previously mentioned processes, it is possible that lower levels of this element could promote progression of colorectal cancer due to weaken antioxidative defense, decreased immune response and disrupted DNA synthesis.

We showed significantly higher copper concentration in malignant tissue as it was the case in the similar study [15]. Copper level was significantly higher in CRC in male subjects while in females this difference showed no significance. Other studies showed elevated Cu levels in various human neoplasm, including breast cancer and lymphoma, many of them focusing on copper’s role in

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angiogenesis, a crucial process for cancer development [15,22–24]. Also, a study

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which was investigating Cu, among other elements, in thyroid gland carcinomas

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and benign thyroid disease, found significantly higher serum Cu levels in thyroid cancer group compared to bening thyroid disease group [25].It has been shown that

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copper salts are endothelial cells migrating stimulators and inducers of fibronectin

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synthesis, a glycoprotein associated with angiogenesis[26,27], so it is evident why copper chelators are one of the anti-angiogenetic agents currently in clinical trials

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[27]. Along with significantly lower Zn levels, higher Cu levels in male subjects could, once again, be a hallmark of CRC invasiveness in men. It was noted in previous studies that serum Cu/Zn ratio is increased in CRC and

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could be a good predictor of certain cancers invasiveness and prognosis [28–30]. We have measured this ratio in CRC and adjacent healthy tissue. To our knowledge, no other study compared this ratio in malignant and healthy tissue. Cu/Zn ratio was significantly higher in malignant tissue in our study and was, also, higher in males and both elderly and middle – aged subjects. In female subjects the

difference in Cu/Zn ration between malignant tissue and healthy tissue was not significant. The role of copper/zinc ratio in CRC tissue requires further investigation, but our results indicate that, like serum Cu/Zn, this ratio could also be a good predictor of cancers aggressiveness and, possibly, poor prognosis. Our study showed significantly higher selenium concentration in malignant

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tissue compared to adjacent healthy tissue, which is in consistence with results of

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similar study [15]. In physiological, low concentrations, Se is essential to humans,

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but it shows toxic features when in higher levels [18]. Study with similar results proposed that higher Se levels in colorectal cancer tissue, despite its protective

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role, could be the result of body’s immune reaction in attempt to induce apoptosis

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of colorectal cancer cells through increasing number of free radicals [15]. On the other hand, the accumulation of oxidative damages was proposed to be one of the

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most important causes of malignant transformation of the cell [31]. Selenium is described as an anticancer agent because of its role in the expression of powerful antioxidants – selenoproteins [32]. Selenoproteins (like glutathione peroxidase 2,

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GPX2) contribute to antioxidant defense mechanisms of the cells by preventing the oxidative damage of the DNA before it happens [32,33]. Thus, rather than the toxic effect of accumulated selenium in the CRC tissue, we think that an attempt to enhance selenoprotein expression is more likely the cause of elevated Se levels in malignant compared to non-malignant tissue.

There were no differences in iron (Fe) and manganese (Mn) concentrations between malignant and healthy intestinal tissue in our study, although other studies showed significantly lower Fe and higher Mn levels in colorectal cancer[7,15,34,35]. The role of Mn and Fe in carcinogenesis can be observed through their role in inhibition of apoptosis, generation of oxygen radicals and

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binding competition among metal ions at chromatin and other molecules. Also,

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animal models revealed association between parenteral iron application with tumor

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growth and higher tumor incidence[15,36–39]. Lack of significant differences of Fe and Mn levels in our study could derive from limited participants and regional

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differences, though their role in colorectal cancer must not be neglected.

Alterations of macroelements in CRC

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Potassium was significantly higher in malignant tissue compared to a healthy adjacent tissue in our study. There are sufficient results regarding potassium levels in colorectal cancer. Our results are consistent with the ones from two other studies

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[15,40] that investigated, among others, potassium tissue levels in CRC compared to healthy colorectal tissue. It is known that tumor cells have high metabolic energy demands to fuel their divison and growth and also one of the proposed emerging hallmarks of cancer is its ability to re-programme energy metabolism [31]. Increased uptake and utilization of glucose via glycolysis have been

documented in many human tumors [31]. It was shown that one of the cardinal glycolytic enzymes, pyruvate kinase M2, was significantly elevated in colorectal cancer samples compared to healthy controls, and that potassium plays one of the key role in functioning of this glycolytic enzyme [41]. Thus, the activity of this enzyme would certainly profit from high potassium content.

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Calcium levels were significantly higher in malignant tissue compared to

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adjacent healthy tissues. Also, this was the only element in our study that showed

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significant difference in malignant tissue levels between males and females with tendency to be higher in men. Most of the samples in our study belonged to

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patients with stage III CRC. Necrosis in cancerous tissue is often present in

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advanced stadiums and it is known that necrotic tissue show accumulation of calcium. It is possible that most of the tumor tissue in our study had necrotic fields

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in biopsy samples and that could be a possible explanation of higher Ca levels in CRC. Also, we believe that higher intracellular calcium concentration could possibly be linked with activation of Ca – dependent enzymes, among others,

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endonucleases, which could lead to damage of the genetic material. It is know that malignant tissue is poor in energy substrates [31]. This could affect Ca – ATPase enzyme system which can lead to intracellular accumulation of this element. On the other hand, high intracellular Ca levels could promote cellular damage by various processes.

In our study Mg levels were higher in colorectal cancer tissue in comparison to healthy adjacent tissue. In vitro studies showed intracellular accumulation of Mg in neoplastic cells [42]. There are also findings which link Mg with angiogenesis, especially with the functions of Vacular Endothelial Growth Factor (VEGF) and endothelial cell migration [43]. Since angiogenesis is one of the hallmarks of

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cancer [31], the relationship between elevated Mg in CRC tissue and this crucial

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process for tumour growth and malignant transformation should be considered.

Alterations of non-essential and toxic elements in CRC

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Chromium (Cr) level was significantly lower in cancerous tissue in comparison

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with healthy controls. There were no differences in malignant tissue Cr levels between males and females or between different age groups. Recent study showed

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higher Cr levels in colorectal cancer tissue compared to a healthy adjacent tissue [7]. On the other hand, Rinaldi et al. noted no difference in Cr levels between cancerous tissue and healthy controls and lower Cr levels in CRC tissue compared

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to adjacent healthy tissue [12]. Because it was proposed that Cr could be involved in processes that are favourable to tumor progression[44,45], its role in colorectal cancer evolution should be carefully investigated in future studies. Very few studies investigated the roles of mercury (Hg), lead (Pb), and arsenic (As) levelsin colorectal cancer. Moreover, there is sufficient data regarding the

differences in levels of the mentioned elements between healthy and malignant colorectal tissue. In our study, mercury levels were significantly higher in cancerous tissue compared to healthy tissue, while there were no significant differences in Pb and As levels between healthy and cancerous tissue of the colon. Mercury exposure was marked as a risk factor for colorectal cancer and it was

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proposed that higher levels of Hg and Pb may promote the occurrence and

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progression of gastrointestinal cancers, which was shown in the recent Chinese

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study [46]. Mercury has an ability to cause major biological damage in humans, firstly by enzyme inhibition, production of free radicals and alteration of tertiary

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and quaternary structures of most proteins [47,48]. Despite well documented

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toxicity of mercury and its role in cardiovascular diseases and neurodegenerative disorders, there is not enough data regarding relations of Hg and colorectal cancer.

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The presence of this element in environment in various forms and variety of exposure to Hg in our region should be considered when interpreting higher mercury levels in colorectal cancer tissue.

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Levels of cadmium (Cd) were significantly lower in malignant tissue compared to healthy controls in our study. Cd is a verified human carcinogen [49]. It is believed that this element is involved in various processes such as generation of ROS, inhibition of apoptosis, inhibition of DNA repair mechanism and gene expression suppression [50]. Levels of Cd

were shown to be elevated in breast and lung cancer tissues compared to healthy controls [51,52], but other studies showed no differences in Cd levels in liver [53]. On the other hand, there are not many studies that investigated its role in carcinogenesis in colorectal cancer. One study [54] found lower Cd levels in colorectal polyps, which are believed to carry higher risk in malignant

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transformation to colorectal cancer [55]. The exact explanation of lower Cd level

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in malignant tissue in our study could be found in the different sample types, small

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cohort, and variable exposure to environmental cadmium in our region. We have observed positive correlations between As and Cd, and As and Hg in

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tumorous tissue, while these correlations were absent in healthy adjacent colon

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tissue. Moreover, we have found mostly negative correlations between these toxic elements and essential elements in both CRC and healthy tissue. These correlations

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should be used in further studies to determine the possible negative effect of mentioned toxic elements and the elements included in antioxidant defense regarding CRC. On the other hand, the correlation between Hg in malignant and

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Hg in healthy tissue was the strongest positive correlation between these tissues. Since the role of As, Cd, and Hg in carcinogenesis is well-documented, and all

of them are considered very important occupational and environmental agents, their mutual relationship and potential agonistic effects in development or progression of CRC should be investigated. Also, since there is sufficient

information regarding Hg and CRC, the observed strongest correlation between Hg in malignant and Hg in healthy tissue should encourage a more detailed analysis of the role of Hg in CRC.

Limitations of the study

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Our study has limitations. First of all, to obtain valid results that reflect

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elemental alterations in CRC compared to healthy colorectal tissue, a much larger

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cohort is required, especially when the patients age and sex are considered as potential causes of different levels of trace elements between malignant and non-

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malignant tissues. Different lifestyle and environmental factors and determinants

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(smoking status, diet habits, physical and occupational activites, housing environment, and stress),must be taken into concern, as it is known that these

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factors have tremendous impact on many (patho)physiological processes in human body. Finally, since oncologic patients have various therapeutic strategies, different drug treatments, along with previously prescribed drugs for different

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conditions, should not be neglected in any research regarding levels of trace elements and the processes they regulate.

Conclusion

We found significant alterations of Na, K, Mg, Ca, Cu, Zn, Se, Mn, Cd, Cr and Hg in malignant tissue of colorectal cancer compared to adjacent healthy bowel tissue. We have found that Cu/Zn tissue ratio was significantly higher in CRC compared to a healthy tissue and that patients with higher CRC stages had also significantly higher ratio. Therefore, we propose further analysis of tissue Cu/Zn

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ratio in CRC because of its potential role as predictor of CRC invasiveness and

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progression. There are differences in lifestyle, dietary intake, pollution and

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exposure to toxic elements among various regions, thus no singular conclusion can be reached for different populations. The people of the Balkans have many lifestyle

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similarities, and, since this is the first such study in our region, we assume that

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results of our study could be a good indicator of elemental alterations in colorectal cancer of Balkan population. Lastly, we think that further studies need to seek an

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answer to a question: “do elemental alterations have role in cancerogenesis or are they a consequence of the disease itself?”

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Author Statements

All authors agree with submitted manuscript and its content.

Funding: This study was supported by the grant No III-41013 from the Ministry of Education, Science and Technological Development, Government of Serbia.

Conflict of Interest: The authors declare no conflict of interest. Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its

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later amendments or comparable ethical standards.

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Informed Consent: Informed consent was obtained from all individual

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participants included in the study.

Skalny A V. Bioelementology as an interdisciplinary integrative approach in

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[1]

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of the gallbladder. J Surg Oncol 2005;91:204–8. doi:10.1002/jso.20306. [29] Khoshdel Z, Naghibalhossaini F, Abdollahi K, Shojaei S, Moradi M, Malekzadeh M. Serum Copper and Zinc Levels Among Iranian Colorectal Cancer Patients. Biol Trace Elem Res 2016;170:294–9. doi:10.1007/s12011015-0483-4.

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Endocrinol Lett 2006;27 Suppl 1:36–9. [52] Martin Mateo MC, Rabadan J, Boustamante J. Comparative analysis of certain metals and tumor markers in bronchopulmonary cancer and colorectal

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cancers. Metals and tumor markers in the neoplastic process. Clin Physiol Biochem 1990;8:261–6.

[53] Mai F-D, Chen B-J, Wu L-C, Li F-Y, Chen W-K. Imaging of single liver tumor cells intoxicated by heavy metals using ToF-SIMS. Appl Surf Sci 2006;252:6809–12. doi:10.1016/J.APSUSC.2006.02.227.

[54] Alimonti A, Bocca B, Lamazza A, Forte G, Rahimi S, Mattei D, et al. A Study on Metals Content in Patients with Colorectal Polyps. J Toxicol Environ Heal Part A 2008;71:342–7. doi:10.1080/15287390701839133. [55] Grady WM, Markowitz SD. The Molecular Pathogenesis of Colorectal

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-p

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Sci 2015;60:762–72. doi:10.1007/s10620-014-3444-4.

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Cancer and Its Potential Application to Colorectal Cancer Screening. Dig Dis

Figure 1. Differences of Cu/Zn ratio between lower (I and II) and higher (III and IV) CRC stages, all subjects

0.14

*

0.12

of

0.08

0.06

ro

Cu/Zn Ratio

0.10

0.04

-p

0.02

0.00 Lower (I and II)

Higher (III and IV)

- indicates statistically significant difference (p<0.05)

Jo

ur na

lP

*

re

CRC Stages

Figure 2. Differences of Cu/Zn ratio between CRC stages in all subjects.

p=0.05 0.14 0.12

0.08 0.06

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Cu/Zn Ratio

0.10

0.04

0.00 I

II

III

Jo

ur na

lP

re

-p

CRC Stage

ro

0.02

IV

Figure 3. Differences of Cu/Zn ratio between CRC stages in female subjects.

*

of ro

0.10

-p

Cu/Zn Ratio

0.15

0.00 I

lP

re

0.05

II

III

- indicates statistically significant difference (p<0.05)

Jo

*

ur na

CRC stage

IV

Table 1. Certified values and measured levels of elements (NIST 1577c, Gaithersburg, MD,USA) Analysed value**

Recovery

(µg/kg)

(µg/kg)

(%)

As

19.3±1.4

20.5±1.1

106.2

Cd

97±1.4

97.9±2.6

100.9

Pb

62.8±1.0

63.3±2.6

100.8

(mg/kg)

(mg//kg)

(%)

Cu

275.2±4.6

271.9±5.7

Fe

197.94±0.65

197.43±5.21

Zn

181.1±1.0

180.9±1.8

Mn

10.46±0.47

10.55±0.25

Cr

53±14

51±2.8

Co

0.3±0.018

0.31±0.016

103.3

Ni

44.5±9.2

52.7±4.3

118.4

Se

2.031±0.045

K

10230±640

Na

2033±64

Ca

131±10

Mg

620±42

ro

99.7

-p

re

The data are presented as means ± standard deviation.

Jo

**

98.8

99.9 100.9 96.2

2.055±0.066

101.2

10540±300

103.0

2011±140

98.9

125±4

95.4

631±19

101.8

lP

Certified value as given by the manufacturer.

ur na

*

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Certified value*

Elements

Table 2. Characteristics of studied population.

Male

29 (49.2%)

Female

30 (50.8%)

Sex

6710 years

Middle-aged

29 (49.2%)(59  7 years)

Older

30 (50.8%)(74  6 years) Colon

37 (62.7 %)

-p

Tumor localization

ro

Mean  SD

Histological type

lP

Synchronous Adenocarcinoma

ur na

I 15 (25.4%) Stages of CRC

II15 (25.4%)

n (%)

III25 (42.4%) IV 4 (6.8%)

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5 (8.5 %)

re

Colorectal junction Rectum

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Age:

16 (27.1 %) 1 (1.7 %)

59 (100%)

Table 3.Differences inelemental levels between tumorous and adjacent healthy tissue of all 59 subjects.

lP

ur na

Jo

SD 331.65 30.29 333.93 370.08 4.80 39.13 18.87 98.29 12.76 53.45 471.78 13.03 0.77 1.74 4.35 43.08 3.73 430.10 1.07

<0.0001*a <0.0001*a <0.0001*a 0.011*a <0.0001*a <0.0001*a <0.001*b 0.821b 0.546b <0.0001*b 0.117a 0.345a 0.118a 0.315a <0.0001*a 0.013*a 0.758a 0.382a 0.231a

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Mean 1879.77 107.60 1780.20 1257.15 19.22 108.40 87.12 152.11 22.46 73.09 276.01 12.50 0.72 1.59 2.73 23.75 4.30 113.71 0.44

p value

ro

SD

-p

Mean

1600.11 340.85 Na[mg/kg] 144.8112 50.14 Mg[mg/kg] 2391.64 611.60 K[mg/kg] 1469.83 581.80 Cu[g/kg] 16.96 3.43 Zn[mg/kg] 165.63 63.90 Se[g/kg] 135.23 95.30 Ca[mg/kg] 156.30 119.28 Mn[g/kg] 23.33 18.97 Fe[mg/kg] 38.57 38.57 Cd[g/kg] 154.95 328.47 Al[g/kg] 10.27 14.32 Pb[g/kg] 0.57 0.24 U[g/kg] 1.36 1.43 As[g/kg] 0.78 0.70 Hg[g/kg] 7.89 19.74 Cr[g/kg] 5.10 19.67 Co[g/kg] 675.42 4870.47 Ni[g/kg] 0.26 0.35 Sn[mg/kg] * - indicates the statistically significant difference a - Paired Samples t-test b - Wilcoxon Signed Ranks Test

Concentration in adjacent healthy tissue

re

Element

Concentration in tumor tissue

Table 4. Differences in elemental levels between CRC and healthy tissue in 29 male subjects.

ur na Jo

SD

1872.16 111.85 1848.52 1278.96 20.53 120.98 87.84 141.72 22.96 77.50 209.28 12.58 0.73 1.80 2.91 24.87 3.96 184.69 0.51

257.30 34.24 333.93 344.56 4.98 43.90 14.55 86.62 15.93 60.42 379.86 11.75 0.92 2.34 3.38 45.36 2.94 604.43 1.41

lP

1580.41 318.86 150.76 61.00 2419.35 627.85 1569.92 642.88 16.68 3.71 175.23 68.57 162.48 123.61 157.62 115.82 24.24 15.93 35.40 39.67 193.06 388.59 12.73 16.13 0.55 0.17 1.15 1.00 0.90 0.89 7.23 16.02 2.45 1.49 43.26 98.32 0.20 0.00 * - indicates the statistically significant difference a - Wilcoxon Signed Ranks Test

Mean

<0.0001*a <0.0001*a <0.0001*a 0.033*a 0.001*a 0.001*a 0.001*a 0.358a 0.496a <0.0001*a 0.614a 0.648a 0.465a 0.070a 0.001*a 0.015*a 0.003*a 0.144a 0.109a

of

Na[mg/kg] Mg[mg/kg] K[mg/kg] Cu[g/kg] Zn[mg/kg] Se[g/kg] Ca[mg/kg] Mn[g/kg] Fe[mg/kg] Cd[g/kg] Al[g/kg] Pb[g/kg] U[g/kg] As[g/kg] Hg[g/kg] Cr[g/kg] Co[g/kg] Ni[g/kg] Sn[mg/kg]

SD

p value

ro

Mean

Concentration in adjacent healthy tissue

-p

Concentration in tumor tissue

re

Element

Table 5. Differences in elemental levels between CRC and healthy tissue in 30 female subjects.

SD 394.91 25.84 302.97 397.95 4.32 29.83 22.50 108.91 15.02 46.38 545.05 14.35 0.66 0.83 5.17 41.58 4.38 85.65 0.59

<0.0001*a <0.0001*a <0.0001*a 0.178a 0.622a <0.0001*a 0.022*a 0.600a 0.943a 0.001*a 0.072a 0.085a 0.138a 0.563a 0.009*a 0.005*a 0.758a 0.686a 0.753a

of

Mean 1887.14 103.49 1714.14 1236.07 17.95 96.23 86.41 162.16 21.99 68.82 340.50 12.43 0.73 1.33 2.56 22.70 4.64 45.10 0.38

re

- indicates the statistically significant difference - Wilcoxon Signed Ranks Test

Jo

a

ur na

*

SD 365.28 36.97 604.97 508.18 3.18 58.70 44.21 124.50 21.75 37.89 259.11 12.13 0.29 1.74 0.42 22.53 27.53 6829.99 0.49

lP

Mean

p value

ro

Na[mg/kg] Mg[mg/kg] K[mg/kg] Cu[g/kg] Zn[mg/kg] Se[g/kg] Ca[mg/kg] Mn[g/kg] Fe[mg/kg] Cd[g/kg] Al[g/kg] Pb[g/kg] U[g/kg] As[g/kg] Hg[g/kg] Cr[g/kg] Co[g/kg] Ni[g/kg] Sn[mg/kg]

1619.15 139.07 2364.85 1373.09 17.23 156.36 108.91 155.03 22.45 41.62 118.11 7.90 0.59 1.54 0.65 7.49 7.65 1286.51 0.33

Concentration in adjacent healthy tissue

-p

Element

Concentration in tumor tissue

Table 6. Differences in elemental levels between CRC and healthy tissue in 30 middle-aged subjects.

*

Mean

SD

1859.33 103.11 1754.03 1218.20 19.47 104.13 84.96 157.30 20.65 71.07 247.36 10.54 0.71 1.66 3.26 26.09 4.69 157.07 0.50

348.83 21.59

- indicates the statistically significant difference - Wilcoxon Signed Ranks Test

Jo

327.23 331.71

0.001*a <0.0001*a <0.0001*a 0.004*a 0.005*a <0.0001*a 0.007*a 0.123a 0.737a <0.0001*a 0.958a 0.733a 0.465a 0.132a 0.001*a 0.001*a 0.033*a 0.753a 0.893a

-p

ro

4.03 30.28 18.28 101.85 8.27 49.72 499.61 9.79 0.91 1.80 4.42 51.50 4.58 592.27 1.41

p value

of

SD 332.15 38.99 624.77 660.06 3.69 53.58 113.77 41.98 7.86 40.49 408.58 13.89 0.23 1.30 0.76 6.87 28.03 6943.71 0.49

ur na

a

Mean 1606.15 138.25 2363.06 1440.73 16.86 164.13 137.19 125.17 21.30 40.38 216.69 10.14 0.55 1.33 0.83 3.86 7.62 1348.26 0.32

re

Na[mg/kg] Mg[mg/kg] K[mg/kg] Cu[g/kg] Zn[mg/kg] Se[g/kg] Ca[mg/kg] Mn[g/kg] Fe[mg/kg] Cd[g/kg] Al[g/kg] Pb[g/kg] U[g/kg] As[g/kg] Hg[g/kg] Cr[g/kg] Co[g/kg] Ni[g/kg] Sn[mg/kg]

Concentration in adjacent healthy tissue

lP

Element

Concentration in tumor tissue

Table 7. Differences in elemental levels between CRC and healthy tissue in 29 older subjects.

SD

1594.26 151.15 2419.26 1497.97 17.04 167.08 133.34 186.38 25.27 36.80 95.26 10.39 0.58 1.37 0.71 11.78 2.65 25.00 0.20

354.62 58.95 607.95 504.63 3.21 73.41 75.22 157.63 25.53 37.21 217.04 14.96 0.24 1.57 0.63 26.48 1.63 0.00 0.00

1899.53 111.94 1805.47 1294.80

318.86 36.67 343.91 405.83 5.48 46.26 19.48 96.18 15.91 57.60 450.03 15.46 0.63 1.70 4.28 33.36 2.68 166.11 0.59

18.97

-p

112.51 89.19 147.09 24.20 75.03 303.69 14.38 0.72 1.51 2.22 21.39 3.92 71.78 0.38

p value 0.001*a <0.0001*a <0.0001*a 0.102a 0.141a 0.001*a 0.002*a 0.221a 0.221a <0.0001*a 0.013*a 0.153a 0.080a 0.390a 0.004*a 0.041*a 0.008*a 0.109a 0.068a

ro

Mean

- indicates the statistically significant difference - Wilcoxon Signed Ranks Test

Jo

a

SD

ur na

*

Mean

re

Na[mg/kg] Mg[mg/kg] K[mg/kg] Cu[g/kg] Zn[mg/kg] Se[g/kg] Ca[mg/kg] Mn[g/kg] Fe[mg/kg] Cd[g/kg] Al[g/kg] Pb[g/kg] U[g/kg] As[g/kg] Hg[g/kg] Cr[g/kg] Co[g/kg] Ni[g/kg] Sn[mg/kg]

Concentration in adjacent healthy tissue

of

Concentration in tumor tissue

lP

Element

Table 10. Differences of Cu/Zn ratio between CRC and healthy tissue in groups of subjects.

All subjects

Cu/Zn ratio [ppb] in CRC tissue (mean  sd) 0.0870.027

Cu/Zn ratio [ppb] in healthy tissue (mean  sd) 0.0690.026

<0.001*a

Males

0.093  0.027

0.65  0.038

<0.001*a

Females

0.081  0.029

0.073  0.031

0.158a

Middle-aged

0.086  0.031

0.064  0.020

0.003*b

Older

0.089  0.026

0.073  0.031

0.017a

Jo

ur na

lP

re

-p

ro

a – Paired samples t – test b – Wilcoxon Signed Rank Test

of

* - indicates the statistically significant difference

p value

Jo

ur na

lP

re

-p

ro

of

Table 9. Significant correlations* of trace elements within CRC and healthy tissue, all subjects. * correlations significant at the level of p<0.01, unless noted otherwise; Spearman’s Rank Correlation a correlations significant at the level of p<0.05; Spearman’s Rank Correlation

Within CRC tissue Correlation coefficient

-0.279a 0.285a 0.377a

K-Mg K-Na K-Se K-Zn

0.745 -0.743 0.581 0.432

0.351 0.390

Na-Se

-0.437 0.292a

ur na

Se-Zn

As-K Cd-K Cd-Mg Cd-Na Cd-Se

Jo

As-Cd As-Hg

Cr-Fe Cr-Na

0.410 -0.291a -0.285a 0.478 0.449

Cu-K Cu-Mg Cu-Mn Cu-Se

K-Mg

lP

Mg-Se Mg-Zn

0.437 0.279a

of

Cu-Na Cu-Se Cu-Zn

Co-Cr Co-Fe

ro

0.468 0.672

Within healthy tissue Correlation coefficient 0.444 0.406 0.388 0.332 0.257a

-p

Cu-K Cu-Mg

Elements Ca-Cr Ca-Cu Ca-Fe Ca-Mg Ca-Mn

0.495

0.600

K-Se K-Zn

0.433 0.800

Mg-Mn Mg-Se Mg-Zn

0.275a 0.427 0.360

Mn-Zn Se-Zn

-0.316 0.353

As-Cu As-Fe As-Mn

-0.300a 0.273a -0.419

Hg-Se

0.293a

re

Elements

-0.300 -0.290a -0.307 0.275a -0.337

0.329a 0.400a

Table 10. Correlations* of elements between CRC and healthy tissue, all subjects. Al

Ca

Cd

Cr

Elements in healthy tissue Cu Fe Hg K Mg

Na

Pb

Se

Zn

0.26 -0.28 0.46a 0.28 -0.29

-0.27 0.29 0.28

0.27 -0.31

0.31

-0.3a

0.33

-0.26 0.28

of

0.59a

0.32 0.31

0.30

correlations are significant at the level of p<0.05, unless noted otherwise; Spearman’s Rank Correlation – correlations are significant at the level of p<0.01; Spearman’s Rank Correlation

*-

Jo

ur na

lP

re

-p

a

Mn

ro

Elements in CRC tissue

Al As Ca Cd Cr Cu Fe Hg K Mn Na Pb Se Zn

As