Introduction

Global mortality due to various types of cancer is increasing and expecting to surge over 13.1 million casualties by 2030 (IARC 2008). Cancer is the third cause of mortality in Iran with an annual death toll of over 30,000 cases. After skin cancer, breast cancer is the most common type of cancer among women and the second leading cause of death after lung cancer (Bray et al. 2004). Breast cancer affects mainly women (99% of cases are women). Generally speaking, breast cancer is the most common cancer in the world in terms of mortality (Barnes and Newman 2007).

In 2016, the WHO reported 519,000 casualties due to breast cancer globally (Szkutnik-Fiedler et al. 2016). In Iran, the number of breast cancer cases was estimated to be 40,000 with 6,000 new cases annually (Otaghvar et al. 2015; Jafari et al. 2018). In recent years, the surge of breast cancer incidence in Iran has led to this cancer as the most common cancer type among women so that more than 30% of cases were under 30 years old (Mousavi et al. 2007). Moreover, in Kurdistan Province, located in the west of Iran, the rate of breast cancer has boosted from 13 new cases in 2000 to 103 cases by 2009, so that is ranked as the fifth common cancer among women.

Several factors including age, family history of breast and uterine cancer, colon and rectum cancer, previous history of breast abnormalities, not getting pregnant or having the first pregnancy after the age of 30, alcohol consumption, smoking, and obesity have been reported to associate with the risk of developing breast cancer (Land et al. 2014; Miller et al. 2018). Environmental pollutants such as trace elements are also suspected to accelerate the incidence of breast cancer. Mortality and morbidity due to air, soil, and water pollution have been reported in the literature (Mohammadi et al. 2020; Hajizadeh et al. 2020; Malakootian et al. 2020; Rehman et al. 2018; Lu et al. 2015). One explanation for these findings could be due to the existence of trace elements and their inextricable association with unhealthy impacts.

Trace elements are the environmental pollutants that play a key role in the occurrence of many diseases among animal and human societies (Mansouri et al. 2012a, b; Rezaei et al. 2021; Farnia et al. 2021). Increasing amounts of trace elements followed by human activities in our environment have had far-reaching clinical effects (Burguera et al. 2002; Rautray et al. 2010). Trace elements and metalloids such as Hg, As, Pb, Cd, and Cr can interfere with human metabolomics, leading to morbidity and even mortality (Rai et al. 2019). They can disturb metabolic functions of the body in different ways. Trace elements may accumulate in various organs (e.g., liver, heart, kidney, and brain) disturbing normal biological functions. Moreover, trace elements can enter the body via the consumption of contaminated food, water, and air (Rajaei et al. 2012; Mansouri et al. 2012c; Rehman et al. 2018). Diseases due to the aforementioned elements include principally respiratory problems, cardiovascular events, kidney diseases, and central nervous system dysfunctions (Manisalidis et al. 2020). Environmental pollutions are linked as an important factor in the occurrence of many cancer types (Lu et al. 2015).

Trace elements are divided into two groups: essential and non-essential metals for the metabolic activities of the human body. Trace elements such as copper (Cu), iron (Fe), and zinc (Zn) are essential elements that play an important role in the body’s metabolic and physiological activities, but in order to avoid their negative side effects, their amounts intake must be controlled (Norouzi et al. 2012; Majnoni et al. 2013; Maleki et al. 2015). For example, Fe can react with peroxides and generate free radicals. The excessive amount of these free radicals can cause damage to DNA, lipids, proteins, and carbohydrates in tissues, leading to oxidative stress (Kawanishi et al. 2002; Leonard et al. 2004). Geraki et al. (2002) found higher iron concentrations in breast cancer patients compared to their controls. Also, Ionescu et al. (2006) obtained significant concentrations of nickel (Ni) in the breast cancer tissue compared to healthy samples.

Metals such as cadmium (Cd), lead (Pb), and chromium (Cr) are non-essential metals that do not play a role in the body’s metabolic activities, and their presence has adverse effects on human health (Rezaei et al. 2019). Some metals (i.e., Cd and Pb) have been reported to be associated with some diseases including breast, cardiovascular, renal, gestational diabetes, hypothyroidism, and thyroid cancer as well as skeletal tissue diseases (Järup 2003; Rezaei et al. 2019, 2021). Since Cd stimulates cell growth, short-term exposure to low concentrations of Cd can damage breast tissue DNA (Roy et al. 2004).

Although the effect of trace elements on breast cancer has been well documented (Roy et al. 2004; Ionescu et al. 2006; Romaniuk et al. 2017; Chanihoon et al. 2021), research on the effect of trace elements on breast cancer in Iran has been largely ignored. Therefore, the main objective of this study was to compare the accumulation of essential and toxic metals (Pb, As, Cd, Ag, Al, Cr, Cu, Fe, Se, and Zn) in breast cancer and non-cancerous breast tissue samples in an Iranian population.

Materials and methods

Study population

This research was a case-control study. The study area of the present study was Kurdistan Province. Kurdistan, also known as Persian Kurdistan, is located at the west of Iran and shares a border with the Arabic Kurdistan region in Iraq. Persian Kurdistan consists of three provinces; one of them is Kurdistan Province with Sanandaj City as its capital. The current population of Kurdistan Province is 1,600,000 (66% in urban versus 34% in rural areas) with an annual rainfall of 500 mm.

Biopsy samples

This study was designed to measure trace elements in breast tissue samples in both cancerous (n = 63) and non-cancerous (n = 63) groups. Initially, individuals attending Kowsar Hospital and its pathology clinics in Sanandaj City during 2019 and 2020 were invited to enroll in the study. Informed consent was obtained before collecting breast tissue samples from participants. The biopsy specimens were then put away in formaldehyde solution with the endorsement of a pathologist. Cancerous patients were selected from 25 to 40 years old women who are at the highest risk of breast cancer development. All patients were newly diagnosed with no specific treatment history. Patients with kidney dysfunctions, anemia, pregnancy, historical gastric surgery, and consumption of zinc, iron, calcium, and selenium supplements were excluded from the study. Controls were selected from women visited by a physician and diagnosed as healthy after pathology examination of breast tissue. Like cancerous tissues, specimens from control tissues were frozen at −20 °C.

Determination of heavy metals

We applied the nitric-perchloric acid digestion method to prepare samples for measuring heavy metals. Approximately 1 g of samples was mixed with 2 mL of ultrapure (HNO3, 65%, Merck, Germany) and 1 mL of perchloric acid (HClO4, 70%, Merck, Germany). The mixture was kept in a water bath (TW12, JULABO GmbH, Germany) at 100°C. After cooling, a mixture was obtained by adding double-distilled water up to 25 mL (18.2 MΩ-cm at 25 °C, Fistreem, WSC044, UK). The prepared solution was measured for Pb, As, Cd, Cr, Ag, Al, Cu, Fe, Se, and Zn using inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7900)(Nozadi et al. 2021). After every four analyses, spikes and control samples were implemented. The recovery of Ag, Al, As, Cd, Cr, Cu, Fe, Pb, Se, and Zn were 98%, 97%, 98%, 96%, 102%, 97%, 101%, and 99%, respectively. The detection of limits for all the elements fell between 0.5 and 2 μg L−1.

Statistical analysis

Descriptive statistics including median and interquartile range were used to report summaries for numerical variables and numbers and percentages for nominal or categorical covariates. Since metal concentrations were usually right-skewed, to tackle this issue, it is a routine practice to standardize concentration levels. Concentrations were log-transformed and then standardized using the mean and interquartile range. To compare the concentrations between the cancer and healthy group, the Mann-Whitney test was used. Potential relationships between trace element concentrations were checked using the Spearman correlation coefficient. Additionally, clustering analysis and principal component analysis (PCA) were used on standardized concentration levels to discover the grouping behavior of trace elements. The results of PCA were used by linear discriminant analysis (LDA) to verify the PCA classification merit in allocating subjects into cancerous or non-cancerous groups. Classification accuracy of subjects was evaluated using the area under the ROC curve (AUC), the true positive rate, and the true negative rate. The effect of studied trace elements on the individual’s risk of breast cancer was evaluated using logistic regression analysis to calculate adjusted and unadjusted risk differences (RDs) between cancerous or non-cancerous groups. RDs were adjusted for age, size of the tumor, and the direction of surgery.

Results

In this study, 126 female participants were recruited and organized in two groups; 63 with breast cancer and 63 served as their controls. The mean age of cancerous patients was 52.29 ± 11.18 (30 to 76 years) and the mean age of controls was 35.02 ± 14.25 (17 to 66 years). Mann-Whitney test revealed a significant mean age difference between the two groups (p < 0.001).

Concentrations of elements

In cancerous group, the general pattern of increasing trace element concentrations was Al < Cu < Zn < Se < Fe < Cr <Pb< As < Cd < Ag. The pattern in non-cancerous group was Al < Zn < Cu < Se < Fe < Cr <Pb< Ag < Cd < As (Table 1). Mann-Whitney test revealed that the levels of As (1.25 vs. 0.30 μg kg−1), Cd (1.1 vs. 0.2 μg kg−1), Cr (17.05 vs. 13.7 μg kg−1), Cu (87.7 vs. 68.6 μg kg−1), Pb (13.1 vs. 6.4 μg kg−1), and Se (58.2 vs. 39.2 μg/L) were significantly higher in cancerous subjects. Zn (102.5 vs 64.55 μg kg−1) was the only trace element with higher levels in healthy subjects. There were no significant differences in Ag (0.55 vs. 0.66 μg kg−1), Al (117 vs. 126.9 μg kg−1), and Fe (28.8 vs. 28.2 μg kg−1) levels between two groups.

Table 1 The levels of trace elements in cancerous and healthy subjects. Data presented as median (25th–75th percentile)
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Relationships between elements

Association between 10 trace elements was investigated using Spearman correlation analysis. Figures 1 and 2 provides a matrix of useful information about trace elements including pairwise scatter plots (lower diagonal), histogram plots (diagonal), and also a measure of association between trace elements (upper diagonal) for both cancerous (Figure 1) and control subjects (Figure 2). Spearman correlation was used to measure the association. Significant correlations were marked with stars: three stars if it was significant at 0.001 (p < 0.001), two stars if it was significant at 0.01 (p < 0.01), and one star if the correlation was significant at nominal 0.05 level. In the cancerous group, correlations between elements were positive but mostly non-significant. Moderate correlations were observed between Al-Cr (r=0.60), As-Cu (r=0.52), and Cu-Se, (r=0.56). In the non-cancerous group, no correlation over 50% (r > 0.50) was observed between elements.

Fig. 1
figure 1

Pairwise correlation analysis of trace elements for cancerous subjects; * significant at 0.05, ** significant at 0.01, ***significant at 0.001

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Fig. 2
figure 2

Pairwise correlation analysis of 10 trace elements for non-cancerous subjects; * significant at 0.05, ** significant at 0.01, *** significant at 0.001

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Cluster 1 consists of As and Fe, cluster 2 Cd-Pb, cluster 3 Zn-Se, cluster 4 Cu, and cluster 5 consists of Ag-Ai-Cr(Figure 3A). For the non-cancerous group, a totally different grouping of elements was observed. There were five clusters but with different elements as those clusters we observed for cancerous patients. Cluster 1 consisted of Ag-Cd, cluster 2 Cu-As, cluster 3 AI-Cr, cluster 4 Pb-Se-Zn, and cluster 5 Fe alone (Figure 3B).

Fig. 3
figure 3

The dendrogram of trace elements resulting for cluster analysis for both cancerous (top panel) and non-cancerous group (bottom panel)

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Moreover, to reduce dimensionality, PCA analysis was used to combine trace elements with similar behavior in one component to evaluate whether it can be used for the classification of individuals as either cancerous or non-cancerous based on their levels of trace elements. PCA revealed that the first five components accounted for 73.4% of the variance (Figure 4). For instance, the first components included 23.7% of the variance and comprise Cd, As, Pb, and Se. Five PCA components were used by LDA to classify subjects into cancerous or non-cancerous groups. LDA showed that AUC was 0.979 with a true positive rate of 93% and a true negative rate of 8%.

Fig. 4
figure 4

ROC curves for predicting cancerous patients and healthy women

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Relationships between elements and breast cancer

Table 2 reports the adjusted and unadjusted risk differences (RDs) for each of the 10 trace elements. Raw RDs show significant effect of the levels of Ag (RD = −0.082; 95% CI, −0.206, −0.041), As (RD = 0.560; 95% CI, 0.419, 0.701), Cd (RD = 0.855; 95% CI, 0.733,0.977), Cr (RD = 0.144; 95% CI, 0.009, 0.279), Pb (RD = 0.557; 95% CI, 0.408, 0.707), Se (RD = 0.172; 95% CI, 0.047, 0.297), and Zn (RD = −0.175; 95% CI, −0.332, −0.017) on the development to breast cancer in women. The positive sign of RD value denotes that compared to controls, the risk of breast cancer increases if the levels of element are increased. The negative sign of RD indicates that compared to controls, in the case of patients, higher levels of elements are contributed to a lower risk of breast cancer. For example, for trace element Ag, the RD value (−0.082) was negative and significant (95% CI does not include zero), which means in cancerous women, the levels of Ag were lower than controls. In other words, elevated levels of Ag contribute to lowering the risk of breast cancer. When the raw RDs adjusted for age, the size of the tumor, and also the direction of surgery, the levels of Ag, Cr, and Zn were no longer significant, but the levels of Cu turned into a significant factor (RD = 0.082; 95% CI, 0.005, 0.159).

Table 2 Risk difference (RD) estimation for elements under logistic regression model
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Discussion

The concentration of trace elements in biological samples and tissues can provide the normal/optimal level of the elements that may be variable depending on age, gender, dietary status, geographic conditions, or genetic characteristics. The results of our study showed an elevated concentration of As, Pb, Cr, Cd, Cu, and Se and lowered levels of Zn in tissues with breast cancer compared to non-cancerous breast tissues. The significant function of trace elements in physiological and metabolic status is well documented. However, the role of these elements in cancer has long been speculated, and many types of research have yielded conflicting results (Kuo et al. 2002). The changes in the concentration of essential trace elements including Fe, Cu, Zn, and Se have been studied in patients with various forms of cancers including breast cancer. However, such investigations are limited in the Iranian populations. Siddiqui et al. (2006) found that compared to healthy subjects, the concentration of Cu, Zn, Fe, and Ca was higher in benign and malignant breast tumor tissues with the significant increase in Cu, Fe, and Ca concentrations in benign tissue and Zn, Fe, and Ca concentrations in the malignant tissue. Ebrahim et al. (2007) reported similar findings for higher levels of Se, Cr, and Zn in breast cancer tissues. Some studies suggest monitoring concentration levels of Cu, Zn, and Se for breast cancer diagnosis, prognosis, and management (Florea and Büsselberg 2011; Riesop et al. 2015). Kuo et al. (2002) proposed a prognostic value of the Zn/Cu ratio for breast cancer diagnosis (Kuo et al. 2002).

Some studies reported a lower blood or serum concentration level of selenium in patients with breast cancer, though Se levels were higher in cancerous tissues compared to normal tissues (Borella et al. 1997; Kuo et al. 2002). The elevated concentration of Se in cancerous tissues is due to this fact that Se can bind easily to proteins and absorb rapidly by growing cells. Ebrahim et al. (2007) in vitro study suggest that selenium can affect endothelial and tumor cells, reducing the main molecules required for angiogenesis (Choi et al. 2019). It has been shown that Se can reinforce strong junctions and strengthen cell-cell attachments in tissues with breast cancer, which is in line with anti-proliferative results of selenium (Chen et al. 2013).

Elevated levels of Cu were detected in cancerous breast tissues compared to non-cancerous tissues (Silva et al. 2009; Raju et al. 2006). Kuo et al. (2002) also found that Cu levels in the serum and tissues of patients with malignant breast cancer were significantly elevated than in benign and normal cases, likely due to being Cu as an effective factor for angiogenesis (Silva et al. 2009). Since Cu can be observed in all living cells and it is an important element in many essential enzymes, it requires some specific events including angiogenesis, immortality, and metastasis (Choi et al. 2019). Although Cu is important for a variety of biochemical functions, it has a potential toxic nature too. Tissue and DNA injury due to oxygen free radicals can play important role in neoplastic formation due to copper accumulation (Raju et al. 2006).

In contrast to our study, some studies have reported elevated concentration levels of Zn in breast cancer tissues (Tapiero and Tew 2003; Ebrahim et al. 2007; Silva et al. 2009) as a result of destroying the protecting effect of Se against cancer progression causing an increase of cancer growth rate. Moreover, Cu accumulation can limit Zn absorption (Kuo et al. 2002). The potential differences between metastases, heterogeneity in treatment protocols, dietary status, and geographic conditions can also affect the trace elements levels in patients with breast cancer.

In this study, we found an elevated concentration of Cr in breast cancer tissues compared to non-cancerous ones. Similar findings have been reported elsewhere (Ionescu et al. 2006; Byrne et al. 2013; Ragab et al. 2014). Despite the metabolic necessity, Cr has been known as a carcinogenic agent. Cr (VI) exposure can lead to phenotypes abnormality due to reactive oxygen species formation and DNA damages. Another possible mechanism for the carcinogenicity of this element is through the suppressing of p53, a protein for tumor suppression (Raju et al. 2006).

In agreement with our study, escalation of lead (Pb) concentration in breast cancer tissues has been reported by other studies (Ionescu et al. 2006; Siddiqui et al. 2006; Ragab et al. 2014; Alatise and Schrauzer 2010). The impaired antioxidant-oxidant balance in patients with breast cancer and tumorous tissue can provide a potential link between oxidative stress and breast cancer pathogenesis (Feng et al. 2012; Gönenç et al. 2006).

Findings of the Cd effects on breast cancer disease are paradoxical; it may potentiate the progression of breast cancer, and also it may inhibit the development of tumors by preventing angiogenesis. Jablonska et al. (2017) reported that elevated concentrations of this metal were more pronounced in small or low-grade tumors. Pacini et al. also showed that exposure of MCF-7 cell line to sub-toxic concentrations of cadmium can lead to a reduction in production and release of anti-cancer factors (Pacini et al. 2009). In contrast, Strumylaitė et al. (2008) showed that Cd concentrations were significantly elevated in malignant breast tissue compared to tumor-free breast tissue. Such contradictory results may partly explain the inconsistency in the epidemiological studies for the association of cadmium exposure and breast cancer risk, emphasizing the need for further studies.

In our study, the focus was to study the distribution as well as the interaction of toxic and essential trace elements. There is good evidence to show that many trace elements have an important role in different biological processes using activation or inhibition of enzymes or using competition with other trace elements for binding sites. Thereby, it is sensible to assume trace elements can affect the carcinogenic process by using complex interactions (Siddiqui et al. 2006).

By clustering analysis, we found several clusters of trace elements in cancerous patients; cluster 1 consists of As and Fe, cluster 2 Cd-Pb, cluster 3 Zn-Se, cluster 4 Cu, and cluster 5 consists of Ag-Al-Cr. Studies to cluster trace elements concentration levels in breast cancer patients are limited. By using hair samples of patients who were at stage III of breast cancer; Cihan et al. (2011) obtained eight clusters of 34 elements which were different clusters of trace elements to what we found in this study. However, there was a similarity in the grouping of Fe and As. We also used PCA to identify potential sources and the grouping of trace elements in normal and cancerous breast tissues. In breast cancer tissues, PC1 showed higher loading for Cd, As, Pb, and Se. These trace elements are mainly associated with environmental exposure, contaminated food, and dietary habits. As expected, PCA results were different for cancerous and normal tissues; therefore, it highlights the main role of certain elements in each group. Pasha et al. (2008) showed higher loadings of Cd, Pb, Co, Cu, Fe, Mg, Mn, and Ni in the benign breast tissues.

The incidence of cancer can be associated with living in polluted areas. Environmental pollution may insert individuals at higher risk of heavy metal exposure. Some studies report the excessive number of heavy metals in the soil, water, and foods of polluted areas (Maleki et al. 2014; Ebrahimi and Ebrahimzadeh 2015; Maleki et al. 2015; Karimyan et al. 2020). For instance, Maleki and Jari (2021) reported that the amount of As and nitrate in the drinking water of Sanandaj city (the capital of Kurdistan province) exceeded the standard limit, suggesting a carcinogenic risk of As. Another study reports high concentration levels of Cr and Pb in vegetables from farms in Sanandaj city that were above the standards threshold limit of the Food and Agriculture Organization (FAO) and also the World Health Organization (WHO)(Maleki et al. 2014). Ebrahimi and Ebrahimzadeh (2015) study also found high levels of Pb, Cr, As, and Hg in 79 (out of the 96) drinking water resources across Kurdistan Province.

The strength of the current study is the application of the ICP-MS method to assess several essential and toxic elements simultaneously in cancerous and non-cancerous breast tissues. This investigation can provide basic information on supporting the effects of essential and toxic elements in breast cancer etiology. However, our study had several limitations. Dietary intake information of patients was not available in our study. Further studies with larger sample sizes are necessary to study more types of tissues and lymph nodes. The involvement of cancerous patients with lymph node metastasis is helpful to assess the potential links between different metals and the involvement of lymph nodes. Also, further studies are needed to establish a relationship between heavy metal concentrations in serum and tissue with nutrient supplementation and patient outcomes with different stages of breast cancer.

Conclusion

In this study, the concentration of different essential and toxic elements in the breast tissue was investigated. The results of our study showed an elevated concentration of As, Cd, Cr, Cu, Pb, and Se and lowered levels of Zn in breast cancer tissues compared to normal ones in the Iranian context. Also, moderate correlations were observed between Al-Cr, As-Cu, and Cu-Se in the cancerous group. In the case of the breast cancer tissues, PC1 showed higher loading for Cd, As, Pb, and Se. Our study provides insights into the possible roles and effects of different elements in breast cancer.