Effects of e-waste exposure on biomarkers of coronary heart disease (CHD) and their associations with level of heavy metals in blood

Excess heavy metals increase the risk of various diseases. Electronic waste (e-waste) is a potential route to heavy metal exposure, and Taizhou is a large e-waste dismantling area in China. In this study, we acquire blood samples from residents living near an e-waste recycling area (exposed group) and other residents in a selected reference area (reference group) for a comparative study in Taizhou in December 2017. Seven heavy metals, including cobalt (Co), nickel (Ni), cadmium (Cd), tin (Sn), copper (Cu), zinc (Zn), and lead (Pb), are quantitatively determined in all blood samples. It is discovered that the levels of Co, Ni, Sn, and Pb in the exposed group are higher than those in the reference group. Additionally, two crucial biomarkers of coronary heart disease (CHD), i.e., troponin (Tn) and myeloperoxidase (MPO), and two biomarkers of oxidative stress, i.e., malondialdehyde (MDA) and 8-isoprostane (8-I), are measured. We discovered that the levels of these indicators in the exposed group are significantly higher than those in the reference group. Meanwhile, both the Spearman correlation and multiple linear regression analysis show that Ni is positively correlated with Tn, MPO, 8-I, and MDA. Hence, we hypothesize that exposure to e-waste increases the risk of CHD and that Ni is an important contributor to the initiation of the disease.


Introduction
Coronary heart disease (CHD) is one of the main types of cardiovascular disease. The main cause of CHD is arteriosclerosis (AS), characterized by thickening of arterial walls and narrowing of hemorrhage, which result in myocardial ischemia and increased oxygen demand (Wirtz and von Kanel 2017). Long-term myocardial ischemia can result in acute symptoms such as heart failure, arrhythmia, cardiac infarction, and angina pectoris. CHD is a disease with a high mortality rate (Liao et al. 2017). In 2008, CHD constituted 30% of global deaths, and the total number of deaths increased to 11 million by 2020 . Troponin (Tn) is a regulatory protein of myocardial contraction that is vital to muscle contraction and relaxation (He et al. 2020). When cardiomyocytes are damaged, Tn is released rapidly into the bloodstream, resulting in elevated levels of Tn in the blood (Chao et al. 1990;Shah et al. 2018). Because of its sensitivity and specificity, Tn can be used as a biomarker of myocardial cell necrosis, and clinical inflammation is regarded as a critical factor in the development of AS. Myeloperoxidase (MPO) is a type of peroxidase formed by the release of neutrophils. It can induce the formation of free radicals, modify low-density lipoprotein, and affect endothelial function, ultimately resulting in unstable AS plaques. MPO levels are assumed to be associated with the severity of CHD (Kurniati et al. 2018;Liu et al. 2012). Elevated levels of oxidative stress (OS), i.e., a series of reaction states caused by an imbalance between oxidation and anti-oxidation in the body, have been shown to be related to the initiation of CHD (Ito et al. 2019). Malondialdehyde (MDA) is a product of the chain reaction of oxygen free radicals, where unsaturated fats in biofilms are attacked and lipid peroxidation that damages tissue cells occurs (Banday et al. 2007). MDA levels are a direct reflection of lipid peroxidation. Meanwhile, 8-isoprostane (8-I) is a lipid peroxidation product catalyzed by arachidonic acid (Patrono et al. 2005;Vassalle et al. 2012). MDA and 8-I are typical biomarkers of OS in the human body.
Heavy metals, which are important raw materials for industrial production, can be released into water, the atmosphere, and soil. Because heavy metals can tolerate decomposition under natural conditions, they persist in the environment (Ra et al. 2013). Heavy metals in the environment can enter the body through various pathways and harm the body through accumulation and biological actions (Ebrahimi et al. 2020). Epidemiological studies have demonstrated relationships between heavy metals and the immune and reproductive systems (Ali et al. 2020;Farkhondeh et al. 2020). Several studies have shown that heavy metals can affect CHD. Longterm exposure to Ni can result in myocardial thickening and hypertrophy (Lou et al. 2013). Chronic accumulation of cobalt (Co) can cause myocardial damage and affect cardiac systolic and diastolic functions (Haga et al. 1996). Meanwhile, heavy metals induce OS, and vascular endothelial dysfunction promotes the formation of AS plaques (Grotto et al. 2010;Vaziri 2008). Exposure to heavy metals is significantly associated with the risk of cardiovascular disease and affects the initiation of CHD (Larsson and Wolk 2016).
Owing to developments in science and technology, many electrical and electronic equipment have been developed. In China, several large electronic waste (e-waste) recycling locations exist, including Guiyu and Taizhou. Many metals are released during e-waste recycling, including mercury (Hg) Co, Ni, Sn, Zn, and Cu Li et al. 2020;Xu et al. 2018;Yu et al. 2017). Nevertheless, to prevent further environmental degradation, manual and scattered e-waste disassembling workshops in Taizhou were closed in 2015. In December 2017, we collected blood samples from residents living near an e-waste dismantling area and a reference area in Taizhou.
The aim of the present study is to investigate the potential effects of e-waste exposure on the risk of CHD, as well as the contribution of heavy metals to the disease by analyzing the concentrations of heavy metals, biomarkers (Tn and MPO) of CHD, and the correlation among them in the blood.

Study areas and sample collection
In the present study, we selected Luqiao District as the exposure site (exposed group), which is a well-known e-waste dismantling area in Taizhou. Huangyan District was selected as the reference site (reference group), which is approximately 20 km from Luqiao and is extremely similar to Luqiao in terms of population, transportation, lifestyle, and socio-economic status. The age of all participants was between 30 and 50 years. Informed consent was obtained for acquiring information such as sex, weight, career history, and residence. The age, height, weight, and body mass index (BMI) of the two groups were similar, as shown in Table 1. The study was approved by the Ethics Review Committee of the Peking University Health Science Center in Beijing, China. After blood collection, all samples were stored at − 80°C in an anticoagulant tube.

Instrumentation and metal determination
Venous whole blood samples were acquired from a vacuum blood collection tube containing K2EDTA. The plasma was preserved at − 20°C after centrifugation (3000 rpm, 2-8°C). Subsequently, 1.5 mL of HNO 3 was added to the sample at room temperature and predigested for 2 h. Next, 0.5 mL of H 2 O 2 was added for further digestion (Microwave Digestion System; mws-2; Bergholt, Germany) and diluted with deionized water to 7 mL. The concentrations of metals in the pretreated samples were determined via inductively coupled plasma mass spectrometry (ELAN DRCII PerkinElmer, USA).The quantitative limit of all target metals was 0.002-1 ng/mL. Seven heavy metals were determined quantitatively.

Biomarkers analyses
Human blood plasma acquired via the procedures above was used as a biomarker. The expression of the following markers of myocardial injury (Tn and MPO) and OS markers (MDA and 8-I) was determined using ELISA based on the manufacturer's instructions.

Statistical analysis
The concentrations of heavy metals and biomarkers are expressed as median values±interquartile range (IQR). Univariate analysis indicated a partial normal distribution for all data in this study. We performed a Spearman correlation analysis (two tailed) to investigate the relationships between different variables. In addition, we used a multiple linear regression model to estimate the relationship between the concentrations of metals and each of Tn, MPO, MDA, and 8-I and used a t-test to evaluate their significance. Statistical significance was set at p < 0.05. The data analysis software used was SPSS version 25.0.  . 1 Concentrations of biomarkers in the blood from reference groups and exposed groups

E-waste exposure elevated levels of biomarkers of CHD and OS in blood
Tn and MPO are markers of inflammation and CHD, respectively. In the present study, we analyzed the levels of two biomarkers in the blood. The results showed that the concentrations of the Tn (576.11 ng/mL) and MPO (183.38 ng/mL) in the exposed group were significantly higher than those (Tn (523.18 ng/mL) and MPO (163.13 ng/mL)) in the reference group (Table 2, Fig. 1). Furthermore, we discovered that the concentrations of the two OS markers (MDA (6.43 pg/mL) and 8-I (1210.94 pg/mL)) in the exposed group were *Significance level of 0.1; **significance level of 0.05; ***significance level of 0.001 significantly higher than those in the reference group (MDA (5.24 pg/mL) and 8-I (1021.72 pg/mL)). CHD is a chronic disease characterized by OS and inflammation. When the body is stimulated by external stimulation, the imbalance of OS results in the formation of significant amounts of lipid products and oxides in the arterial walls, which induces an immune response and an inflammatory reaction (Abraham and Marchuk 2014). Several studies have shown that MPO mediates the formation of OS and inflammatory reactions and is a significant inflammatory marker (Love et al. 2017;Zhang et al. 2020). Moreover, the oxidative modification of MPO causes the formation of arterial plaques, and the level of MPO is closely associated with CHD (Gorudko et al. 2012). During the initiation of CHD, inflammatory cell infiltration and OS can cause oxidative damage to cardiomyocytes and other tissues. When cardiomyocytes are slightly damaged, membrane rupture occurs, and Tn is released from the cardiomyocytes into the peripheral blood (Mair et al. 1994). Our results showed that e-waste exposure elevated the blood levels of CHD and OS biomarkers, i.e., Tn, MPO, 8-I, and MDA, which indicates an elevated risk of CHD in residents living near e-waste recycling sites, and that OS is involved in the process.

Associations between biomarkers and heavy metal exposure
The results of the levels of the seven heavy metals in the blood samples are shown in Table 3. Compared with the reference group, the concentrations of Co, Ni, and Sn in the exposed group were significantly higher than those in the reference group (Table 3).
The results of Spearman correlation analysis showed that the concentrations of Co, Ni, and Sn were significantly positively correlated in the exposed group, indicating that the concentrations of heavy metals in the human blood were affected by exposed e-waste. To investigate the contributions of heavy metals to the risk of CHD, we analyzed the relationships between altered heavy metals and biomarkers of CHD and OS using Spearman correlation analysis and multiple linear regression. In the exposed group, the Spearman correlation analysis showed that the concentration of Ni was positively correlated with the concentrations of Tn, MPO, MDA, and 8-I. Meanwhile, the concentration of Co was positively correlated with 8-I and MDA, and 8-I and MPO were positively correlated with Sn (Table 4, Fig. 2). In addition, the linear regression analysis showed that Ni was correlated with Tn, MPO, and MDA, whereas Co was correlated with Tn (Table 5). In the reference group, both the Spearman correlation and linear regression analyses did not indicate any association between heavy metals and the biomarkers of CHD and OS (Tables 4 and 5).
Meanwhile, the results showed that exposure to heavy metals can affect OS in the body, as well as increase the corresponding levels of CHD markers. The results showed a significant correlation among MPO, 8-I, and MDA (Table 4).
Epidemiological and toxicological studies have shown that exposure to heavy metals is associated with cardiovascular Table 5 Multiple linear regression analysis for analyzing associations between hormones and metals in participants of reference and exposed groups  (Houston 2011). Heavy metals contribute significantly to CHD (Myong et al. 2014). Heavy metal-rich monocytes accumulate in the aortic wall through blood circulation and induce endothelial cell injury and apoptosis (Kukongviriyapan et al. 2014). Excessive amounts of heavy metals cause cardiotoxicity through apoptosis and DNA damage (Owumi et al. 2020). Metals can cause OS and inflammation, increase blood pressure, and affect thrombosis (Valera et al. 2012). After exposure to heavy metals, the body produces an inflammatory response, resulting in elevated levels of MPO, which increases the risk of CHD (Reisgen et al. 2020;Xiao et al. 2019). In a study pertaining to the cardiac toxicity of heavy metals, it was discovered that heavy metals can cause an increase in serum Tn levels (Ali et al. 2020). Our results showed that e-waste exposure elevated the blood levels of Ni and that Ni was positively correlated with the biomarkers of CHD and OS, including Tn, MPO, MDA, and 8-I. Ni can promote LDL oxidation and affect AS (Wu et al. 2015). Furthermore, Ni damages the vascular endothelium and alters vasoconstriction/vasodilation, which are important symptoms of CHD (Cuevas et al. 2010). Occupational Ni exposure can increase lipid peroxidation and decrease antioxidant levels (Kalahasthi et al. 2006). Moreover, OS is vital to the pathogenesis of CHD (Yang et al. 2019). Elevated levels Ni was positively correlated with the level of these indicators in exposed group of MDA and 8-I suggest that exposure to heavy metals can result in lipid peroxidation, which can subsequently cause cell and tissue damage and hence the formation of plaques in the arteries. Based on the findings above, we infer that Ni can increase the risk of CHD and OS.

Conclusion
In summary, although almost all e-waste workshops have been closed for 2 years, the effect of exposure to heavy metals persists in the associated areas. Compared with the reference group, the levels of Co, Ni, and Sn in the exposed group were higher. This indicates that e-waste exposure can result in the accumulation of heavy metals and increase the level of heavy metals in the body. Therefore, the problem of heavy metal exposure mediated by e-waste must be investigated comprehensively.
In addition, it was discovered some critical biomarkers of CHD and OS in the blood were elevated in residents living near the e-waste recycling area. Furthermore, a significant correlation was discovered among Ni, Tn, MPO, MDA, and 8-I, indicating that e-waste exposure increased the risk of CHD and that Ni is an important contributor to the initiation of the disease.
Author contribution Wang and Xue is mainly responsible for the data analysis and drafts the manuscript. Li and Wei curated the study and oversaw the coordination. All authors were involved this study and read and approved the final manuscript.
Funding This work was supported by grants from the National Natural Science Foundation of China (21477119).
Data availability Available on request.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Review Committee of Peking University Health Science Center in Beijing, China.

Consent to participate Not applicable.
Consent for publication All of the authors have reviewed and approved the manuscript for publication.

Competing interests
The authors declare no competing interests.