The Association Between Arsenic Levels and Oxidative Stress in Myocardial Infarction: A Case–Control Study

Cardiovascular diseases (CVDs) are known as the first causes of death throughout the world, and mainly myocardial infarction (MI), lead to 7.4 million deaths annually. Atherosclerosis is the major underlying cause of most CVDs. However, exposure to heavy metals, among other factors, deserves further attention as a risk factor for CVDs. This study was designed to evaluate the levels of arsenic (Ars) in myocardial infarction (MI) patients and healthy individuals as well as assess the association between the incidence of MI and Ars, total antioxidant capacity (TAC), and oxidative stress. This case–control study was conducted among patients with MI (n = 164) and normal individuals (n = 61) at Shafa Hospital in Kerman, Iran. Patients were classified into two groups, including coronary artery blocks above 50% (CAB > 50%, n = 83) and coronary artery blocks less than 50% (CAB < 50%, n = 83) based on their angiography findings. The demographic characteristics, clinical history, biochemical parameters, and serum Ars and TAC levels were evaluated. In the present study, both CAB groups had significantly reduced levels of TAC compared with the control. Furthermore, TAC was lower in the CAB > %50 group compared to the CAB < %50 group. Ars levels were significantly higher in both CAB groups compared with the control. There was a significant positive relationship between CAB and Ars, BG, HbA1c, urea, creatinine, TG, TC, and LDL-c, as well as a negative relationship between HDL-c and TAC. Moreover, TAC levels showed a significant inverse correlation with Ars, HbA1c, and creatinine, and a positive correlation with HDL-c. As risk factors, Ars, hs-CRP, TG, TC, and LDL-c enhance the severity of the disease, and HDL-c and TAC decrease the disease severity. Moreover, ROC curve analysis revealed that the highest AUC for the CAB > %50 (AUC = 78.29), and cytotoxic levels for both CAB groups (Ars ≥ 0.105 ppm), and no significant differences were found between the two groups. Our findings suggest that Ars at ≥ 0.105 ppm is able to increase the risk of MI through the increased OS and decreased TAC.


Introduction
Cardiovascular disease (CVD) is a group of cardiovascular system disorders whose primary cause of the development is generally atherosclerosis [1]. Atherosclerosis refers to the proliferation of intimal smooth muscle and the accumulation of WBCs, inflammatory cells, fatty materials, and extracellular matrix in the intima of arteries that can result in an increased arterial stiffness and blood flow restrictions [2]. An abrupt and persistent blocked blood flow to the heart can result in acute coronary syndrome (ACS), a severe cardiac condition that is usually accompanied by acute myocardial infarction (AMI) [3,4]. As reported by WHO, AMI is the major cause of fatality across the world, accounting for nearly 9 million deaths per year [3,5]. Genetic predisposition, dyslipidemia, diabetes mellitus (DM), cigarette smoking, an unhealthy lifestyle, hypertension (HTN), etc. have been identified as well-known risk factors for CVDs which eventually trigger oxidative stress (OS) and inflammation as crucial mechanisms leading to myocardial damage [6].
The term OS refers to an imbalance between reactive oxygen species (ROS) formation and antioxidant defense systems. Under normal physiological circumstances, ROS levels are tightly modulated by enzymatic and non-enzymatic antioxidant defense systems [7,8]. In fact, OS can prompt the production and enhance the activity of pro-oxidant enzymatic systems that are known to be considerably implicated in the development of atherosclerosis [6]. Among them, NADPH oxidases and myeloperoxidase as the main sources of ROS such as hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 -) play a critical role in the progression of atherosclerotic disease [9,10].
OS is able to alteration of major biological macromolecules such as DNA, proteins, and lipids [11]. The oxidative alteration of low-density lipoprotein (LDL-c) in the arterial intima is thought to be an important initial step in atherosclerosis [12]. Investigations recommend that oxidized LDL (ox-LDL-c) is realized and taken up by macrophages. The transformation of macrophages into lipid-laden cells is a key feature in the formation of the lipid layer and ultimately plaque erosion within the arteries [13][14][15][16]. During atherogenesis, antioxidants have been shown to inhibit the formation of ox-LDL-c. Besides, the activity of serum paraoxonase (PON), which protects LDL from oxidation [17].
In the past few decades, trace elements and environmental exposures to toxic heavy metals such as arsenic, copper, cadmium, mercury, and lead have been associated with public health concerns across the world due to their predictable detrimental effects on human health [18][19][20][21][22][23]. Alongside the other issues, arsenic-contaminated drinking water has become a main universal public health consideration [24]. Human exposure to arsenic (Ars) has been confirmed to be associated with severe clinical manifestations including various cancer types and a broad range of non-cancerous diseases such as CVDs [24][25][26][27][28]. Investigations has indicated a positive relationship between long-term arsenic exposure and an elevated risk of CVD-correlated morbidity/mortality [24].
Experimental studies have demonstrated that Ars enhances ROS production [29,30]. In humans and animals, arsenic exposure induces the formation of ROS/RNS [31]. In addition, experimental studies exploring arsenic toxicity indicate that oxidized lipids during atherogenesis are responsible for the generation of numerous bioactive molecules (e.g., MDA, 4-HNE, and isoprostanes) [32,33]. Moreover, Ars has been found to prompt atherosclerosis by enhancement in the TGF-β [34] and inflammatory factors such as TNF-α, MCP-1, and IL-6 [35]. Despite the well-known role of Ars as a carcinogen, the associations between environmental toxic metals such as Ars and increased risk of CVDs remain less well characterized. Due to the dramatic increase in CVDs and comorbidities, we decided to assess the serum level of Ars and their relationship with OS and TAC levels in MI patients for the first time.

Study Design and Participants
This case-control study was conducted on MI patients at Shafa Hospital in Kerman, Iran. Based on the angiographic results, these patients were divided into two groups: those with coronary artery blockage greater than 50% (CAB > %50, n = 83) and those with less than 50% (CAB < %50, n = 81). This research includes 61 healthy people with no history of heart disease or myocardial infarction. In this study, all Helsinki principles were observed and approved by the ethics committee of Kerman University of Medical Sciences (IR.KMU.AH.REC.1398.117).

Samples and Data Collection
Following recorded consent, participants' demographic knowledge such as education, residence, smoking, kind of water, rice, fish, and oil consumed, family history of cardiovascular and infectious diseases, DM, and HTN were investigated. Then, 10 ml of peripheral blood was drawn from each subject, serum was separated by centrifugation (3500 rpm for 10 min), serum was aliquot, and stored at − 70 °C until analysis.

Total Antioxidant Capacity (TAC) Assay
The ferric reducing antioxidant power (FRAP) assay was conducted according to previous research, which measures ferric (Fe 3+ ) to ferrous (Fe 2+ ) ability at pH 3.5. At 593 nm, a FRAP reagent and serum combination gives a blue color and the sample TAC levels were calculated using the standard curve FeSO 4 .7H 2 O [36].

Arsenic Level Assay
To summarize, serum was diluted with a 0.1% Triton™ X-100 and 0.2% (v/v) nitric acid solution. The total amount of Ars was measured by FAAS (Varian SpectrAA-600 Flame Atomic Absorption Spectrometer) with a hollow cathode lamp and a standard specifically for Ars. The serum concentration of Ars was measured in ppm.

Statistical Analysis
Qualitative data were represented as a percentage (%), whereas numerical data were presented as mean standard error of mean (M ± SEM). The Kolmogorov-Smirnov test and, based on it, parametric or non-parametric-related test were employed. Chi-square/exact Fisher's test, one-way ANOVA/Kruskal-Wallis with post-hoc Tukey/Mann-Whitney U tests were used to compare demographic characteristics and experimental information between groups, respectively. The odds ratio (OR) of disease (MI) was assessed using a multivariate logistic regression test with adjustments for age, gender, residence, smoking, and BMI. Furthermore, in logistic regression, the data were represented in quarters, with the 1st quarter serving as a reference. The correlation coefficient, Spearman's rho, was used to demonstrate the relationships between variables. The diagnostic efficacy of Ars for screening MI (CAB < %50 & CAB > %50) was evaluated by receiver operating characteristic (ROC) curves with a 95% CI and area under the curve (AUC). Cut-off values were determined for Ars based on Youden index. For the statistical analysis, SPSS software version 23.0 (SPSS Inc, Chicago, IL) was used, and charts were prepared by Graph-Pad Prism software (8.4.3). P values < 0.05 were regarded as statistically significant.

Demographic Characteristics of the Study Participants
The demographic characteristics of participants are represented in Table 1. Age was higher in CAB > %50 (p = 0.014) than in control, and males were increased in CAB < %50 (p = 0.007) and CAB > %50 (p < 0.001) than control. Both CAB groups had significantly higher levels of education (< diploma), fish consumption per month (2-4 times), village residence, solid oil consumption, tube water consumption, infectious disease, family history of heart disease DM, and HTN compared to the control group. Disease history significantly is higher in CAB > %50 than in CAB < %50 and control groups. Indeed, CAB > %50 significantly increased Iranian rice consumption and decreased Indian rice consumption.

TAC and Arsenic Levels
In Fig. 1, TAC and Ars levels were represented. Based on this, Ars was boosted in CAB < %50 and CAB > %50 (P < 0.001), and TAC was reduced in CAB < %50 and CAB

Correlation Coefficient Analysis
A Spearman's correlation analysis was performed to determine the relationship between variables, and the outcomes are displayed in Table 3. The correlation values range between − 0.480 and 0.840. CAB has a significant positive relationship with Ars, BG, HbA 1 c, TG, TC, LDLc, age, urea, and creatinine, and a negative relationship with HDL-c and TAC. TAC had a significant inverse correlation with Ars, HbA 1 c, creatinine, and age, and a direct correlation with HDL-c. HbA 1 c and TC were significantly related to Ars. hs-CRP had a significant direct correlation with HbA 1 c and urea. LDL-c had a significantly positive relationship with waist, HbA 1 c, BG, Creatinine, TG, TC, and HDL-c had a significantly negative relationship with creatinine, TG, and TC. HbA 1 c had a significantly direct correlation with age, waist, BMI, and BG. TG has a significant positive correlation with creatinine, creatinine has a positive correlation with urea, age has a positive correlation with urea and BG, and BMI has a positive correlation with waist circumference.

Logistic regression
Adjusted logistic regression was used to identify the severity of the MI (CAB < %50 & CAB > %50), and the data are described in Table 4.

Discussion
Despite compelling evidence on the association of environmental exposure to Ars and an enhanced risk of atherosclerosis [37][38][39], the mechanisms of Ars-mediated increase in atherosclerosis remain to be elucidated. We hypothesized that there is an association between Ars serum and TAC levels in MI patients. In this study, we demonstrate that Ars levels were increased in CAB < %50 and CAB > %50, and also TAC levels were reduced in CAB < %50 and CAB > %50 in comparison with the control group. Besides, CABs demonstrated a positive relationship with Ars, TG, TC, LDL-c, BG, HbA 1 c, age, urea, and creatinine, as well as a Apart from drinking water, dietary sources such as rice are likely to be the considerable sources of daily exposure to low levels of arsenic in populations. In our study, Iranian rice consumption was higher among participants with CAB > %50. Our findings of higher rice consumption in participants with CAB > %50 are consistent with findings from the UK. Xu et al., suggested that exposure to Ars from rice could be considered a potential risk factor for CVDs. The authors revealed that Ars exposure above 0.3 μg/person/day is associated with an increased CVD risk. However, several variables such as ethnicity distribution, ethnicity-related gene polymorphisms, the type of rice, etc. may affect the Parameters are presented as mean ± SEM. one-way ANOVA/Kruskal-Wallis with post-hoc Tukey/Mann-Whitney U tests were used to analyze data. a: Comparison of the control and CAB < %50 groups; b: Comparison of the control and CAB > %50 groups; and c: Comparison of the CAB < %50 and CAB > %50 groups. The significance level is as: * p < 0.05, ** p < 0.01, *** p < 0.001 TG Triglyceride, TC total cholesterol, HDL-c high-density lipoprotein-cholesterol, LDL-c low-density lipoprotein-cholesterol, HbA 1 C glycated hemoglobin, BG blood glucose, hs-CRP high-sensitivity C-reactive Protein, EF % Ejection fraction percentage susceptibility to Ars intoxication [40]. Uncertainties also exist in assessing dietary sources of Ars. Sobel et al., found no association between rice consumption and several relevant markers of subclinical CVD in a multiethnic survey. However, they recommended that probable Ars-specific pathways in CVD progress may be related to the other biomarkers including E-selectin and ICAM-1 that were investigated in their previous studies [41].
It is now generally believed that atherosclerosis is a multifactorial arterial disease. Previous studies have reported that long-term, low-to-moderate-level exposure to Ars caused mild injury to the blood vessels. It is also found that exposure to Ars via drinking water raises the risk for some CVDs such as ischemic heart disease [42], MI [43], HTN [44] and peripheral arterial disorder [42]. In the present study, we found a significant correlation between Ars serum levels and CABs. In fact, Ars serum levels were raised in the patients with CAB < %50 and CAB > %50. In line with this report, Wang and coworkers observed that living in areas with Ars contamination of groundwater (> 0.35 mg/mL) has been associated with an increased prevalence of CVDs [45]. In another survey conducted on 10,910 participants participated in the Health Effects of Ars Longitudinal Study (HEALS). Data indicated that there was an affirmative relationship between low-to-moderate levels of exposure to Ars via drinking water and high pulse pressure [46], an independent predictor of vascular stiffness which is accompanied by an enhanced risk of atherosclerosis [47].
Animal experimental studies have demonstrated that oxidized lipids play a key role in all processes of atherogenesis, and immunizing animal models of atherosclerosis with the protein adducts of the ox-LDL products reduces the generation of atherosclerotic plaque [48,49]. Oxidized lipids are capable of generating numerous bioactive molecules [24]. In humans, high blood levels of Ars are associated with elevated levels of OS, and compromised function of the antioxidant defense system [50]. In our study, the TAC assay has shown that TAC in the groups with CAB < %50 and CAB > %50 was remarkably reduced in comparison with that of the control group. Indeed, TAC decline was greater in the CAB > %50 group than in the CAB < %50 group. Consistent with our study, in a study performed by Muthumani et al., chronic exposure to Ars at a concentration of 5 mg/kg significantly decreased the levels of enzymatic and nonenzymatic antioxidants in rats [51]. Similarly, Liang et al., reported that Ars has the ability to induce cardiotoxicity and dyslipidemia in rats. Their results showed that treatment with Crocin mitigated cardiotoxicity triggered by Ars trioxide via Keap1-Nrf2/HO-1 pathway, thereby resulting in a reduction in ROS production and an increase in glutathione, glutathione S-transferase (GST), glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) levels [52].
According to the results of the current study, CAB groups showed a significant positive correlation with BG, HbA 1 c, TG, LDL-c, TC, Ars and age as well as a negative correlation with HDL-c and TAC. In the context of risk factors, Ars, hs-CRP, TG, TC, and LDL-c enhanced clinical severity of the disease, while TAC and HDL-c reduced clinical severity of the disease. The present study is somewhat consistent with the previous investigation on the biological gradient between atherosclerosis in the carotid and chronic exposure to Ars. The authors reported that prolonged exposure to arsenic prompted ischemic heart disease in adult residents in an arseniasis-endemic region of southwestern Taiwan in a dose-response manner. In fact, a dose-response relationship was found between carotid atherosclerosis indices (CAIs) and serum levels of TG, TC, and LDL-c. The biological gradient even persisted statically significant after adjusting for age, gender, alcohol consumption, DM, smoking status, HTN, and serum levels of TC and LDL-c [53]. Earlier reports from Aranda et al., recommended that there is a connection between the seafood intake and its estimated amount of heavy metals (Ars, Hg, Cd, Pb) and the lipid panel and lipid peroxidation. In fact, the authors reported that an enhancement in the assessed levels of Ars and Hg in seafood was related to an enhancement in the levels of LDL-c, non-HDL-c, APO B/A ratio, and ox-LDL. As it is Fig. 1 Comparison of TAC and Arsenic in study groups. Parameters are presented as mean ± SEM and one-way ANOVA/ Kruskal-Wallis with post-hoc Tukey/Mann-Whitney U tests were used to analyze data. The significance level is as: *p < 0.05, **p < 0.01, ***p < 0.001 Table 3 Spearman's correlation Spearman's rho test was performed to examine the correlation between continuous variables and the significance is as follows:  known, ox-LDL would lead to lipid peroxidation even at low concentrations [54].
It is now recognized that OS and inflammation play an important role in a number of chronic diseases including atherosclerosis, obesity, and DM [55]. Our data showed that TAC level had a significant inverse association with, Ars, HbA 1 c, and age, and a positive relationship with HDL-c. Similarly, Çapaş et al., reported that the levels of serum TG, uric acid, and HbA 1 c in diabetic subjects higher and inverse association with TAC and HbA 1 c in diabetics [56]. In a study conducted in Taiwan, Ars was correlated negatively with the antioxidant capacity levels in plasma and positively with ROS levels. Additionally, they suggested that steady OS in peripheral blood may be associated with the incidence of cancers and atherosclerotic CVD in populations who have been exposed to arsenic for a long time [57]. Furthermore, in line with the present results, an increase in the serum levels of TC, TG, LDL-c, VLDL and concomitant reduction in the serum level of HDL were observed in rats exposed to Ars [58]. In fact hypertriglyceridemia in cooperation with abnormally low levels of HDL-c observed is one the most common atherogenic profile of lipid metabolism in CADs [59]. Additionally, in this investigate, ROC analysis was performed to assess the power of the diagnostic value of serum Ars level and to identify its optimal cut-off value to predict MI incidence in the CAB groups. The results revealed that for the group with CAB > %50, the AUC and the best cut-off values for ROC curve analysis were 78.29 and > 0.105 ppm, respectively, and for the group with CAB < %50 were 74.01 and > 0.105 ppm, respectively. Although the AUC value for the group with CAB > %50 was higher than that of the group with CAB < %50. Our results suggest that in cases of Ars exposure, Ars at concentrations ≥ 0.105 ppm may enhance the incidence and development of MI. This finding from the current study may be somehow consistent with the findings from the previous study which revealed the undesirable effect of Ars exposure on diastolic function. Karakulak et al., reported that the average blood Ars concentration in the Ars-exposed workers and in the controls were 42 and 3.75 µg/dL, respectively. They also reported that the AUC and the cut-off point for blood Ars concentration in the Ars-exposed workers were 83.7 and 45.3 µg/dL, respectively. Furthermore, the authors showed that there is relationship among Ars exposure, diastolic function, and OS, and concluded that the Ars exposure may unfavorably affect diastolic function via OS [60].
In this study, MI patients were classified into two groups, CAB > %50 and CAB < %50, based on angiographic findings. Related parameters were measured, analyzed, and subsequently compared to the controls. However, our study had some limitations. First, due to the design of a cross-sectional study, the individuals are not followed over time, and cardiovascular end-points are not assessed. Second, this study was primarily designed to investigate the association between the serum levels of Ars and TAC in MI patients with MI. However, the concentration of Ars in water, rice, and soil was not measured. Third, due to the affinity of Ars to sulfhydryl groups, it is incorporated into hair and fingernails. So, Ars measurement in these samples is considered to be a better scale to show long-standing arsenic toxicity compared to Ars blood measurement.

Conclusion
In summary, our present evidence is that Ars levels in serum were increased in the patients with CAB < %50 and CAB > %50, and is inversely related to the TAC. The results of the present study also indicated that CAB had a positive association with the serum levels of Ars, HbA 1 c, TG, TC, LDL-c, urea, and creatinine, as well as a negative association with HDL-c and TAC. As risk factors, Ars at ≥ 0.105 ppm is able to increase the risk of MI through the increased OS and decreased TAC. The findings of the present research may inspire investigators to implement further studies respecting probable prognostic and antioxidant-related therapeutic clinical applications. Fig. 2 The ROC curve and the best cut-off for Ars in CAB < %50 and CAB > %50