Evaluation of oxidative stress and pro-inflammatory cytokines in occupationally cadmium exposed workers

Abstract

BACKGROUND:

Cadmium (Cd) exposure in environmental and occupational settings is a major public health concern. Cd exposure is associated with the production of free radicle and reactive oxygen species. The aim of the present study is to evaluate the effect of occupational exposure to Cd on oxidative stress and pro-inflammatory biomarkers in the workers.

METHODS:

100 occupationally exposed individuals working in the metal handicraft industry and welding industry were recruited from the industrial area of Jodhpur, Rajasthan. Blood Cd levels were estimated using atomic absorption spectroscopy. Serum Total Antioxidant Capacity (TAC), Catalase (CAT), Superoxide Dismutase (SOD), and Malondialdehyde (MDA) levels were measured by colorimetric method to assess oxidative status and serum IL-6 and TNF-α were measured by ELISA to assess inflammatory status.

RESULTS:

The median Cd levels in the study population was 2.40μg/L, with welders having significantly higher Cd levels than metal handicraft workers. Among the oxidative stress markers, TAC and CAT were significantly lower, while MDA was significantly higher in subjects with high Cd levels. The Cd levels showed a significant negative and positive correlation with TAC and MDA, respectively. IL- 6 and TNF-α did not show a significant difference between the study groups, but both had an inverse correlation with antioxidant enzymes.

CONCLUSION:

Occupational exposure to even low levels of Cd may result in oxidative stress in workers primarily via decrease in antioxidant enzymes and increasing lipid peroxidation. Increased oxidative stress in turn may result in immune cell activation which may result in increased concentration of pro-inflammatory cytokine in the exposed workers.

Keywords

Cadmium oxidative stress antioxidants inflammation

1 Introduction

Cadmium (Cd) is a non-biodegradable heavy metal with a variety of industrial applications [1]. It is widely used in many industrial settings like batteries, metal plating, pigments, plastics, and alloy industries. The main route of exposure to Cd in humans includes inhalation, ingestion of contaminated food and water. Cd may induce adverse health effects even at very low concentrations, ranging from acute to chronic harm, to various organ systems including gastrointestinal, renal, nervous and reproductive system [2]. Due to known toxic effects, Cd and its compounds are classified as Group 1 carcinogens by the International Agency for Research on Cancer (IARC) [3].

The exact mechanism by which Cd induces toxicity is unclear; however, studies have demonstrated that exposure to Cd may cause the formation of reactive oxygen species (ROS), resulting in oxidative injury to cells [4]. Cd primarily binds to the thiol-containing antioxidant enzymes, which in turn results in decreased antioxidant levels in the body, thus further aggravating oxidative stress in the cells [5]. Inflammation and oxidative stress are a closely related phenomenon where the former can induce the later and vice versa. The ROS generated in response to Cd exposure may activate the phosphorylating enzymes, which further activates the transcription factors associated with inflammation pathways [1]. Also, oxidative stress in itself may result in immune imbalance, causing tissue damage [6].

Cd is also known to have a differential effect on the immune system. In-vivo and in vitro studies suggest an immunomodulatory and immunosuppressive effect of Cd [7, 8]. However, the exact impact of Cd on the human immune system is still unclear. Therefore, the purpose of this study was to evaluate the effect of occupational Cd exposure on oxidative stress and pro-inflammatory cytokines.

2 Materials and methods

2.1 Study subjects

Study subjects (n = 100) were recruited from factories in Jodhpur, involved in metal handicraft (n = 49) and welding works (n = 51), after obtaining ethical clearance from the Institutional Ethics Committee (IEC) AIIMS, Jodhpur. The work processes of the subject involve hammering, cutting, grinding, scraping of metal sheets and welding via manual arc welding with a covered electrode (MMA). The metal artist, in particular, are also involved in engraving, embossing, carving designs and painting on white metal and other metal sheets. Information regarding medical illness, source of drinking water, smoking, alcohol consumption and food habits were collected utilizing a self-developed questionnaire. Subjects were defined as smokers when they smoked >100 cigarettes in a lifetime and currently smoked, while non-smokers were defined as subjects who had not smoked >100 cigarettes in their lifetime and did not smoke at present [9]. Subjects with any history of medical illness such as diabetes, hypertension, cancer, autoimmune or any other immune disorder or using any anti-inflammatory drugs were excluded from the study. Informed consent was obtained from all participants prior to participation.

2.2 Sample collection

6 mL of venous blood was collected from each subject under aseptic conditions. 3 mL of blood was collected in EDTA containing tube for Cd estimation while 3 mL blood was taken in a plain tube without any anticoagulant for serum separation. Blood collected in plain tubes were kept for 10 minutes and later centrifuged at 1500 rpm for 10 minutes. Separated serum was transferred to Eppendorf tubes and stored at –80°C for further use.

2.3 Blood cadmium levels (BCd)

Whole blood was used for determination of Cd levels in Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) with Zeeman correction in an ICE 3500 system (Thermo Fischer Scientific, Waltham, MA, USA). A three-point calibration curve of 0.125 ppb, 0.250 ppb and 0.500 ppb were prepared from serial dilution of Cd stock solution (1000 ppm). The absorbance of the standard solutions after correction for blank was measured and plotted against their corresponding concentrations. The acceptable fit for the calibration curve was taken as >95%. Supra pure-grade 65% HNO₃ (w/v) (Merck, Darmstadt, Germany), ammonium dihydrogen orthophosphate (Thermo Fisher), and Triton X-100 (Sigma) were used to prepare matrix modifier. Samples were diluted (1:20) with a matrix modifier and 0.2% HNO₃. CLIN Check Whole Blood Control Levels I, II, and III (Recipe, Germany), containing 1.23μg/L, 2.88μg/L, 6.32μg/L of Cd respectively, were used for quality control. Absorbance was measured at 228.8 nm. Results are expressed as μg/L. Chemicals and reagents used for the analysis were of analytical grade, and proper aseptic measures were maintained. All cups, pipettes, tubes and beakers used for the analysis were soaked in 0.5% HNO₃ overnight then dried and used to assure no metal contamination.

2.4 Detection of serum levels of pro-inflammatory cytokines

Serum levels of pro-inflammatory cytokines IL-6 and TNF-α were measured with an Enzyme- Linked Immunosorbent Assay (ELISA), according to the manufacturer’s instructions (Krishgen Biosystems, Mumbai, India). In short, samples were added to 96 microwell plates pre-coated with the monoclonal antibody. Biotinylated antibody solutions were added and plates were incubated at 37°C for respective durations. Then,the plates were washed, streptavidin-HRP solution was added and incubated for one hour. Furthermore, the plates were rewashed for multiple times, TMB substrate was added and incubated for 10–15 minutes prior to addition of stop solution. The microplates were fed onto an Eon Biotek multiplate (14021320) ELISA plate reader and wavelength was set at 450 nm. Values were estimated by Gen5 software (version: 2.05.5) using five-point calibration curves. Results are expressed as pg/ml.

2.5 Estimation of oxidative status markers

Antioxidant status was assessed by estimating serum levels of Total Antioxidant Capacity (TAC), Superoxide Dismutase (SOD) and Catalase (CAT). TAC levels were determined by Colorimetric Assay Kit (ABTS, Enzyme Method) (Elabscience) as per the manufacturer’s instruction. SOD levels were estimated by the colorimetric method using the hydroxylamine method (Elabscience) and CAT levels were estimated by the colorimetric method using the readily available kit as per manufacturer’s instruction (Elabscience). CAT and SOD activity are expressed as U/mL while TAC is expressed as mmol/L. Serum MDA was measured as a lipid peroxidation marker using readily available ELISA kit (Krishgen Biosystems, India) following the manufacturer’s instruction and is expressed as nmol/L.

2.6 Statistical analysis

Statistical analysis was carried out in GraphPad Prism version 8.0 software. A p-value <0.05 was considered to be statistically significant. Test for normality by Shapiro-Wilk test showed a non- parametric distribution of data; therefore, continuous variables are represented as median and IQR while categorical data are presented as N (%). The study participants were, divided into two categories according to the median of BCd (2.40μg/L) of the total study population as High Cd group (>2.40μg/L) and Low Cd group (<2.40μg/L). Mann-Whitney U test was used to determine the significant difference between two groups. Spearman’s correlation analysis was used to assess the correlation between BCd levels, oxidative stress markers and inflammatory markers.

3 Results

The general characteristics of the study population are summarized in Table 1. No significant difference between age, BMI, years of exposure and blood pressure among welders and metal handicraft workers were observed. However, a high BMI in welders as compared to metal handicraft workers was present. In addition, smoking history was comparable between the groups. Furthermore, a significantly increased BCd levels were observed in welders when compared to handicraft workers.

Table 1
General characteristics of the study population

Total Welders Metal handicraft workers p value

(n = 100) (n = 51) (n = 49)

AGE (years) 25.50 (16.25) 25 (12.25) 28 (17.75) 0.60

BMI (kg/m²) 21.38 (5.88) 22.06 (5.38) 20.41 (7.13) 0.57

SBP (mm Hg) 120 (14.7) 120 (15) 120 (17.5) 0.76

DBP (mm Hg) 80 (20) 80 (17) 80 (20) 0.16

Education

No education 20 (20) 16 (31.37) 21 (42.85) 0.23

Primary school level 65 (65) 20 (39.21) 18 (36.73) 0.79

Middle school or higher 15 (15) 15 (29.41) 10 (20.40) 0.32

Years of Exposure 5 (8) 5 (6) 4.00 (10.75) 0.81

Smokers 52 (52) 31 (62) 21 (42.85) 0.09

BCd (μg/L) (Blood Cadmium) 2.40 (1.42) 2.80 (1.4) 1.86 (0.64) <0.0001

	Total	Welders	Metal handicraft workers	p value
AGE (years)	25.50 (16.25)	25 (12.25)	28 (17.75)	0.60
BMI (kg/m²)	21.38 (5.88)	22.06 (5.38)	20.41 (7.13)	0.57
SBP (mm Hg)	120 (14.7)	120 (15)	120 (17.5)	0.76
DBP (mm Hg)	80 (20)	80 (17)	80 (20)	0.16
Education
No education	20 (20)	16 (31.37)	21 (42.85)	0.23
Primary school level	65 (65)	20 (39.21)	18 (36.73)	0.79
Middle school or higher	15 (15)	15 (29.41)	10 (20.40)	0.32
Years of Exposure	5 (8)	5 (6)	4.00 (10.75)	0.81
Smokers	52 (52)	31 (62)	21 (42.85)	0.09
BCd (μg/L) (Blood Cadmium)	2.40 (1.42)	2.80 (1.4)	1.86 (0.64)	<0.0001

Data are presented as median (IQR) and if categorical then N (%). p value <0.05 is considered significant.

Mean±SD of BCd level among the study participants were 2.48±1.20μg/L. Study subjects were further divided into two groups on the basis of median BCd as high Cd group (>2.40μg/L) and low Cd group (<2.40μg/L). Oxidative stress markers were compared between the groups. Lipid peroxidation marker, MDA, was significantly higher in the high Cd group. Among the markers of antioxidative status, TAC, CAT and SOD levels were lower in high Cd group, although SOD levels were not significantly lower. Among the pro-inflammatory cytokines, IL-6, and TNF-α levels were higher in the high Cd group as compared to the low Cd group, although the difference was not statistically significant (Table 2).

Table 2

Oxidative stress and pro-inflammatory cytokines in the high and low Cd group

	High Cd group	Low Cd group	p value
	(n = 46)	(n = 54)
MDA (nmol/mL)	6.95 (12.38)	1.63 (0.98)	<0.001
TAC (mmol/L)	0.77 (0.68)	1.26 (0.84)	0.01
SOD (U/mL)	19.04 (7.37)	19.46 (13.62)	0.96
CAT (U/mL)	82.19 (58.09)	94.15 (34.18)	0.05
IL-6 (pg/mL)	5.50 (19.85)	4.68 (12.85)	0.69
TNF-α (pg/mL)	29.86 (276.65)	20.69 (374.88)	0.90

Data are presented as median (IQR). MDA; Malondialdehyde, SOD; Superoxide Dismutase, TAC; Total Antioxidant Capacity, CAT; Catalase.

Furthermore, when the study subjects were stratified according to median years of exposure (5 years) of the total study population as <5 years and >5 years, TAC levels were significantly lower in the subjects exposed for >5 years of duration, whereas, CAT and SOD levels were lower but not statistically significant. Additionally, MDA levels were observed to be higher in >5 years of exposure group but the difference was statistically insignificant. No significant difference was observed in IL-6 and TNF-α levels between the two groups (Table 3). Subgroup analysis on the basis of smoking status revealed no significant difference in the serum levels of the oxidative stress marker MDA between smokers and non-smokers; however, smokers were found to have higher MDA levels as compared to non-smokers. Moreover, among the antioxidant markers, SOD levels were significantly lower in smokers, whereas TAC and CAT were lower in smokers, although statistically insignificant (Table 4).

Table 3

Oxidative stress biomarkers and pro-inflammatory cytokines in subjects as per year of exposure

	>5 years	<5 years	p value
	(n = 45)	(n = 55)
MDA (nmol/mL)	1.82 (1.33)	1.77 (1.71)	0.98
TAC (mmol/L)	0.88 (0.7)	1.27 (0.84)	0.01
SOD (U/mL)	19.22 (12.42)	19.28 (8.51)	0.46
CAT (U/mL)	84.39 (49.49)	92.93 (51.02)	0.10
IL-6 (pg/mL)	4.94 (22.27)	4.68 (10.06)	0.85
TNF-α (pg/mL)	29.27 (205.13)	33.93 (457.62)	0.53

MDA; Malondialdehyde, SOD; Superoxide Dismutase, TAC; Total Antioxidant Capacity, CAT; Catalase.

Table 4

Comparison of oxidative stress and pro-inflammatory cytokines among smokers and non-smokers

	Smokers	Non-smokers	p value
	(n = 52)	(n = 48)
MDA (nmol/mL)	2.13 (1.87)	1.63 (0.97)	0.13
TAC (mmol/L)	0.86 (0.85)	1.20 (0.70)	0.33
SOD (U/mL)	17.67 (10.69)	20.78 (8.63)	0.02
CAT (U/mL)	85.85 (38.21)	86.63 (41.43)	0.84
IL-6 (pg/mL)	5.04 (30.66)	4.77 (4.38)	0.45
TNF-α (pg/mL)	47.95 (276.22)	24.35 (382.76)	0.69

Spearman’s correlation analysis was performed to find the association of oxidative stress markers and proinflammatory cytokines with BCd levels. While a significant positive correlation was observed between BCd and MDA (r = 0.200; p = 0.05), there was a significant negative correlation of BCd with TAC (r = –0.245; p = 0.01). The SOD (r = –0.07; p = 0.42) and CAT (r = –0.11; p = 0.26) did not correlate with BCd. Among the proinflammatory cytokines, while IL-6 had a significant positive correlation with BCd (r = 0.217; p = 0.02), TNF-α did not correlate with BCd. Furthermore, IL-6 showed a significant negative correlation with TAC (r = –0.23; p = 0.02) and SOD (r = –0.201; p = 0.03), and TNF-α showed a significant negative correlation with TAC (r = –0.200; p = 0.05) and CAT (r = –0.20; p = 0.05). The MDA levels did not correlate with either pro-inflammatory cytokines (Table 5).

Table 5

Spearman’s correlation between oxidative stress markers and pro-inflammatory cytokines with BCd levels

	BCd	IL-6	TNF-α
MDA (nmol/mL)	r = 0.20;	r = 0.04;	r = 0.14;
	p = 0.05	p = 0.64	p = 0.17
TAC (mmol/L)	r = –0.24;	r = –0.23;	r = –0.20;
	p = 0.01^*	p = 0.02^*	p = 0.05
SOD (U/mL)	r = –0.07;	r = –0.20;	r = –0.10;
	p = 0.42	p = 0.03^*	p = 0.29
CAT (U/mL)	r = –0.11;	r = –0.12;	r = –0.20;
	p = 0.26	p = 0.21	p = 0.05
IL-6 (pg/mL)	r = 0.21;	–	r = 0.15;
	p = 0.02^*	p = 0.122
TNF-α (pg/mL)	r = 0.05;	r = 0.15;	–
	p = 0.61	p = 0.122

r: Spearman’s correlation coefficient. ^*p < 0.05.

4 Discussion

Cd is a heavy metal with known adverse health effects. According to Agency for Toxic Substances and Disease Registry (ATSDR) 2015, individuals involved in ore smelting operations, drying of cadmium pigments, soldering or welding of cadmium-containing ores, metal coatings, Cadmium containing batteries, phosphate fertilizers, electroplating, alloy production, and petroleum refining are at greater risk for toxic effects of this metal [10]. Studies in and around Jodhpur, Rajasthan have reported the presence of higher concentrations of heavy metals such as Lead (Pb), Cadmium (Cd), Zinc (Zn), Mercury (Hg), and Iron (Fe) [11, 12].

In the current study, median BCd levels in the exposed workers (2.40μg/L) were found to be significantly higher than the permissible range of the World Health Organization (WHO) (0.03–0.12μg/L) [13]. Welders were found to have significantly higher B-Cd than metal handicraft workers which may be attributed to increased inhalation of Cd rich welding fumes in welders. Similar findings with increased Cd levels were reported by Poreba et al. in-steel plant workers [14]. Zhou et al. reported increased Cd levels in smelters was associated with dyslipidemia [15]. However, Indian data on occupational exposure to Cd is sparse, Moitra et al. reported urinary Cd levels of 5.8μg/dL in jewellery makers of Kolkata, West Bengal [16]. Recently in our previous study, we reported increased Cd and Pb levels in occupationally exposed workers of Jodhpur [17].

Cd is known to induce oxidative stress by causing an imbalance between antioxidants and ROS. Our study observed a significant decrease in TAC along with a decreasing trend in the levels of two principle antioxidant enzymes SOD and CAT. Furthermore, an inverse association between BCd and antioxidant levels (TAC) warrants the fact that Cd exposure may result in oxidative stress primarily by decreasing the antioxidants. Similar findings of decreased CAT levels were reported by Conterato et al. in which painters reported to have significantly higher B- Cd levels [18]. Other experimental, as well as occupational studies, reported similar findings of low SOD level in the presence of Cd [19 –22]. However, contradictory results with a positive correlation of B-Cd with SOD and CAT levels have also been reported by some authors, suggestive of a compensatory increase in antioxidant levels in response to Cd-induced oxidative injury [23, 24].

Among the oxidative stress markers, malondialdehyde (MDA) was significantly increased in the high Cd group and also correlated positively with BCd levels. MDA is formed as a byproduct of primary and secondary lipid peroxidation. Increased levels of MDA are highly toxic for cells, and it may induce alterations in enzyme activity, mutations and DNA damage [25]. Increased MDA and decreased antioxidant enzymes draw a parallel for Cd-induced oxidative stress in Cd exposed workers. These findings were similar to previously published studies, where increased MDA levels were observed in workers exposed to welding fumes containing heavy metals like Cd [26 –29].

ROS-induced oxidative stress may result in dysfunction of immune cells. Moreover, immune cells also generate ROS at the site of inflammation which may further accentuate the oxidative stress. The formation of ROS additionally signals for the expression of proinflammatory genes [30]. In our study, we found that serum levels of IL-6 and TNF-α were increased in the high Cd group compared to the low Cd group, although the difference observed was not statistically significant. However, there was a significant positive correlation of IL-6 with Cd levels. Among the oxidative stress biomarkers, a significant negative correlation with the antioxidant enzyme with IL-6 and TNF-α was observed. These findings are suggestive of Cd-induced enhancement in expression of proinflammatory cytokines by immune cells resulting in oxidative cell injury. Apart from activating nuclear factor kappa B (NF-κB), ROS is involved in activation of other factors such as activator protein 1 (AP-1), the hypoxia-inducible factor (HIF-1α), peroxisome proliferator activator receptor gamma (PPAR-γ), β-catenin/Wnt, and nuclear factor-like 2 (Nrf-2) either directly or indirectly. All these factors are involved in the production of adhesion molecules and chemokines and cytokines [31].

Our study has limitations that should be taken into account. Urinary Cd, a marker for long term Cd exposure, would have been a better indicator as blood Cd represents recent exposure to the metal. Measurements of Cd in the workplace were not performed, that would have further affirmed occupational exposure to Cd. Additionally, the evaluation of other oxidative stress markers in a larger sample size would give a clearer image of the adverse effects of Cd in workers occupationally exposed to this toxic metal. Furthermore, welders are also known to exposed to other metals including chromium, cobalt, lead, mercury, nickel, zinc, antimony, and vanadium with different concentrations that could also have impacts on inducing oxidative stress. Unfortunately, due to funding constraints, we were not able to analyze those parameters and we thus acknowledge this as a limitation.

5 Conclusion

Occupational exposure to Cd even at a low concentration is associated with oxidative stress and inflammation. Cd exposure correlates with alteration in oxidative stress markers like MDA and TAC. While the antioxidant enzymes are reduced, oxidative damage marker MDA increases with higher Cd exposure. Furthermore, Cd may also induce the production of pro-inflammatory cytokines like IL-6 and TNF-α in a dose-dependent manner. Finally, there is an inverse relationship of IL-6 with oxidative stress markers, the mechanisms of which need to be explored in future studies.

Footnotes

Acknowledgments

The authors would like to thank the All India Institute of Medical Sciences, Jodhpur for funding the study.

Conflict of interest

The authors declare that they have no conflict of interest.

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