Strontium and antimony serum levels in healthy individuals living in high‐ and low‐risk areas of esophageal cancer

Abstract Background It has been shown there is an upward trend for strontium (Sr) and antimony (Sb) levels from low‐risk (LR) to high‐risk (HR) areas of etiology of esophageal cancer in water, soil, and grains grown in Golestan province. In the present study, the serum levels of Sr and Sb were determined in healthy individuals living in these areas. Methods This cross‐sectional study was performed on fasting blood serum of adult healthy individuals collected by cluster sampling. Subjects were divided into two groups, those living in either HR or LR areas. Strontium and antimony serum levels were measured using a graphite furnace atomic absorption spectroscopy. Results A total of 200 volunteers were enrolled from which 96 persons (48%) and 104 persons (52%) were from either HR or LR areas, respectively. The sex distribution was 40.9% male and 59.1% female, and the average age of enrolled people was 50.9 years. The average strontium levels were 30.44 ± 4.05 and 30.29 ± 3.74 μg/L in LR and HR, respectively. It also has been shown the average antimony levels were 15.21 ± 3.40, 14.81 ± 3.17, 15.13 ± 3.62, and 15.07 ± 3.62 μg/L in LR, HR, urban, and rural populations, respectively. Conclusion The serum levels of strontium and antimony were not significantly different in healthy adults living in high‐ and low‐risk areas of esophageal cancer. However, the average antimony serum levels in Golestan Province were above the reference interval in different countries.

years, but there is an increasing trend of breast cancer in Golestan Province. 7,8 Strontium is the fifteenth abundant element on the earth that includes 0.02 to 0.03 percent of the earth's crust. Strontium is found in air, soil, and water and also is present in several pollutants due to human activities such as industry and agriculture. 9 Strontium has a bone-seeking property. It has been shown that some compounds containing strontium, such as strontium ranelate, stimulate osteoblasts to produce new bone as well as inhibit of the osteoclasts and finally prevent the reuptake of the bone. 10 Some experimental studies have designed to investigate the anabolic mechanism of strontium in bone formation which may shed light into the carcinogenic properties of strontium. 11,12 The carcinogenic potential of strontium was strengthened considering its physiochemical properties which are similar to calcium and could increase ERK and activate RAS signaling pathways. 13,14 Antimony is an element with atomic number 51 that is located in row five and Group 15 of the periodic table. This element is the same group with elements such as arsenic and bismuth. Mainly in nature presents the trivalent Sb (III) and pentavalent Sb (IV) form of antimony. 15 Exposure to antimony can occur via natural sources and industrial activities. 16 Most toxic compounds of Sb are the antimony potassium tartrate that is in toxicity similar to arsenic oxide and caused alike diseases. 17 Antimony containing compounds has been used successfully in the treatment of leishmaniasis for over half a century. 18 The antiproliferative property of organo-antimony against human breast and lung cancer cells is shown in the study of Polychronis and co-authors. They also revealed that this compound act better than cisplatin. 19 On the other hand, exposure to low dose of antimony has been shown to increase proliferation and migration of prostate cancer cells. 20 The results of the study of Keshavarzi et al 6,7 showed the positive association between antimony and strontium with esophageal cancer incidence in Golestan Province.
In the present cross-sectional study, we have evaluated strontium and antimony levels in the serum of individuals living in either HR or LR regions of Golestan Province. Previous studies have also shown that Sb and Sr levels in soil, grains, sediments, and loess deposits have exceeded the permissible limit in this region. This trespassed has been an increasing trend from the LR to the HR of the province. 6,7 Sb and Sr enter the human body via the soil-plant-food chain. [21][22][23][24] Hence, we hypothesized that by measuring the serum levels of these elements in people living in either high-risk or low-risk areas of esophageal cancer, we might find a correlation between higher serum levels of these elements and higher incidence of esophageal cancer.

| Population study
This study was descriptive, analytical, and cross-sectional. Two hundred people were enrolled, 100 individuals from the HR area (kalale) and 100 people from the LR area (Kordkuy) of EC in Golestan F I G U R E 1 Strontium standard curve (The x-axis and y-axis represents the standard concentration and absorption, respectively) Province. Healthy subjects and adult (over 30 years old) of both sexes were enrolled, and patients with cancer and blood hemolysis were omitted from the study.
Reagent: All reagents were analytical grade. Double-distilled water (DDW) was used throughout the experiment. Other materials used in this study include concentrated hydrochloric acid, acetonitrile, concentrated nitric acid, ascorbic acid, Triton X-100, Antifoam, and palladium chloride. The working solutions were prepared, including a diluent solution, modifier solution, and nitric acid 30% (for washing and soaking).
To build a diluent solution, we solved the amount of 1.0556 gr ascorbic acid in DDW and also 50 µL Triton X-100 added into it, then were well mixed and after that added enough DDW to bring the volume up to 12.5 mL and finally, add to it 50 µL antifoams. To construct a palladium chloride solution, an amount of 10 mg of palladium chloride was added in 100 µL of concentrated hydrochloric acid, and then it was well mixed afterward bring the volume up to 10 ml with DDW.

| Strontium measurement
Stock standard solution of strontium nitrate with a concentration of 1000 ppm was used. This solution was diluted, and series of working standard concentrations of 5, 10, 20, 30, and 40 μg/L were prepared. Furthermore, the standard solution without strontium nitrate was used as a calibration blank. A standard curve was constructed as shown in Figure 1.
To match the standard matrix with the serum sample matrix, we used acetonitrile for all dilutions. All the standard and frozen serum samples (after thawing) were mixed with acetonitrile at a ratio of 1:5.
Acetonitrile deproteinizes the serum samples.
Atomic absorption spectrometer equipped with a graphite furnace (YOUNG LIN AAS 8010 model) with strontium Hollow Cathode Lamp was used to analyze the samples. The parameter requirements of the device for the determination of strontium are given in Table 1.
For the analysis of test data, we used the Student t test analysis.

| Antimony measurement
A stock standard solution 1000 ppm from antimony chloride was used.
This solution was diluted and made the series of working standard concentrations of 5, 10, 20, 30, and 40 μg/L as well as 0.0 ppm standard as a calibration blank. The standard curve is shown in Figure 2.
For evaluation of antimony, to match the standard matrix with serum sample matrix, nitric acid 10% for all dilutions. Each of the standard concentrations and also the frozen serum samples (after thawing) were mixed with diluent solution and a modifier solution with a volume ratio of 2:1:1.
Atomic absorption spectrometer equipped with a graphite furnace (YOUNG LIN AAS 8010 model) and without pieces auto-sampler was used that which controls with workstation of AAS 8000 Atomic Absorption Spectroscopy software. Antimony hollow cathode lamp was used for reading samples. The parameter requirements of the device for the determination of antimony are given in

| RE SULTS
In the present study, a total of 200 people were enrolled in which 96 individuals (48%) from the HR area and 104 individuals (52%) from LR area also 40.9% men and 59.1% female. The mean age of participants was 50.9 with 14-year standard deviation.
There was no significant difference between the serum con- There was no significant difference between serum concentrations of Sb in the age-group 1 (14.59 ± 3.14 μg/L) compared with the agegroup 2 (15.59 ± 3.55 μg/L).
To perform further analysis on the studied population, we evaluated West, East, and the whole province in terms of variables such as age, sex, and urban or rural residence, which results showed no significant differences in each of the groups (Table 2).

| D ISCUSS I ON
Due to the potential of pentavalent antimony compounds for the treatment of Leishmaniasis, as well as indirect evidence for the carcinogenicity of strontium in drinking water, extensive studies have been conducted on the biological role of antimony and strontium. 7,18,[25][26][27][28] Several studies found that trace elements such as antimony and strontium in drinking water are associated with cancer. [29][30][31] Also, the anti-cancer properties of some antimony compounds have been tested on the cell lines in vitro. [19][20]32,33 For these reasons, the importance of evaluating the serum levels of Sb and Sr in cancers, especially esophageal cancer, has been highlighted.
Keshavarzi et al 6,7 in a study with the aim of evaluation drinking water quality in the HR area for EC in Golestan Province indicated that villages in the HR areas of the province do not have a good-quality drinking water and the levels of Sr and Sb have been exceeded the permissible limit in drinking water, soil, grain, loess deposits, and sediments of Golestan Province and showed the growing trend from LR to HR. Some studies have shown that F I G U R E 2 Antimony standard curve (The x-axis and y-axis represents the standard concentration and absorption, respectively) Sr is present in the number of pollutants due to human activities (industry, agriculture, and transport) and enters into the body via a soil-plant-food chain. [21][22][23][24] On the other hand, other studies have revealed that the high urinary levels of these elements, which are due to prolonged exposure and make individuals susceptible to various diseases. 32,[34][35][36][37] Also, Makris et al 25   Note: Data are presented as mean ± SD (number of participants/percent of participants).
Abbreviations: HR, high-risk areas; LR, low-risk areas; WP, Whole of province. study investigated the reaction between glutathione and potassium antimony tartrate and concluded that the 4-hour exposure of Sb (III) to erythrocytes resulted in an increase in extracellular glutathione concentration and glutathione efflux. It is also concluded that about 98 percent of the total body content of strontium is implanted in bone tissue, 11,12 and almost 17 percent of strontium be excreted through the urine, which is the main way to dispose of strontium from the body. 34 Since the risk of EC increases proportional with increasing of age, 2 and older people are considered to have longer exposure to strontium and/or antimony, subjects in this study were divided into two age-groups (above 52 years old and below 52 years old).
Nevertheless, there was no significant difference between group 1 and group 2, while EC and the incidence of morbidity increase proportionally with age (such that in the seventh decade of life reaches its peak). 1 50 Chen has been inferred that strontium similar to calcium can activate ER through the CaR, and its effects are alike to estrogen. 35 Since there is an increasing trend of breast cancer in Golestan, 7,8 therefore, we assumed might be different levels of Sr and Sb in the serum of men and women. Contrary to our assumption, the difference between men and women in West, East, and the whole of the province was not significant. This could indicate that probably occupational contamination has no significant role in increasing the serum levels of Sb and Sr.
On the other hand, it is important to note that in studies con-

| CON CLUS IONS
The serum levels of strontium and antimony were not significantly different in healthy adults living in high-and low-risk areas of esophageal cancer. However, the average antimony serum levels in Golestan Province were above the reference interval in different countries.