Risk assessment in mining-based industrial workers by immunological parameters as copper toxicity markers
Rajani Ganpatrao Tumane, Nirmalendu Nath, Aqueel Khan
Department of Biochemistry, RTM Nagpur University, Nagpur, Maharashtra, India
|Date of Web Publication||15-Apr-2019|
Dr. Nirmalendu Nath
Department of Biochemistry, RTM Nagpur University, Lit Premises, Amravati Road, Nagpur - 440 033, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Manifestation of certain health morbidity in copper dust–exposed subjects impels us to obtain a cogent view regarding the implicate relationship of immunoglobulin on health risk assessment in them. We present here immunological profile of copper pit worker and office employees residing in the vicinity. Rationale of this study is to delineate the risk factors involved for copper toxicity. Materials and Methods: Copper mine workers (n = 87) were selected from a copper mine at Malanjkhand. Blood metal concentration and immunological profile such as IgG, IgM, IgA, and IgE were analyzed from these subjects. Data analysis was carried out using proper statistical tools. Results: Chronically copper dust–exposed miners (N = 71) and office employees (N = 47) exhibited significantly higher contents of copper (P < 0.0001) when compared with normal control. Serum IgG and IgA were found to be elevated significantly (P < 0.0001) in them when compared with both office employees and normal control. Contrarily, significant decrease in serum IgM was observed in both the groups when compared with normal control (P < 0.0001). Serum IgE was found to be elevated more significantly only in miner when compared with normal control. Copper exhibited significant positive Pearson's correlation coefficient with IgE, IgG, and IgA (r = 0.39; r = 0.28; r = 0.21) but negative correlation (r = −0.39) with IgM. Odds ratio analysis validated that elevated levels of IgE in miner and decrease in levels of IgM in both groups were truly affected by increase in copper levels from normal to abnormal. Conclusion: Miners are prone to morbidity such as type 2 diabetes and respiratory discomfort (asthma and hypersensitivity) since imbalance in both IgM and IgE is known to be associated with such morbidity. Immunopathy observed in chronically exposed miners could be attributed to copper toxicity in them.
Keywords: Copper, epidemiology, immunoglobulins, metal mine toxicity, occupational exposure and potential markers
|How to cite this article:|
Tumane RG, Nath N, Khan A. Risk assessment in mining-based industrial workers by immunological parameters as copper toxicity markers. Indian J Occup Environ Med 2019;23:21-7
This is a cross-sectional study of mine workers (n = 87) in the age group of 20–60 years selected from copper mine from Malanjkhand area of Madhya Pradhesh (India). These subjects were further subdivided into two groups and classified according to their period of exposure: 0–10 years as Mild or Miner-I (n = 16) and >11–30 years as chronically exposed miners or Miner-II (n = 71). Acute exposed group (Miner-I) was purposely excluded from this study since they were found to show lesser epidemiological, biochemical, and hematological aberration. For the purpose of comparison, a separate group of subject (n = 30) was chosen from nonmining area such as Nagpur region, which served as normal control after age and sex match. Office workers (n = 47) not working in the mine but residing in the same mining were chosen as experimental control after age and sex match.
Blood sample collection
Venous blood samples were collected from all subjects using aseptic conditions. To minimize the possibility of blood sample contamination, workers were instructed to report for collection before the start of shift; 5 mL sterile syringes (metal-free) were used for collection of blood; 3 mL of whole blood was collected in sterile tube for determination of metal concentration in blood. The remaining blood samples were allowed to clot and centrifuged at 1000 rpm for 5 min. Aliquots of serum samples were allowed to freeze immediately and stored at −40°C in accordance with accepted procedures. Thaw serum samples were then used for determination of immunoglobulins (IgG, IgM, IgA, and IgE) with standard procedure.
Determination of serum IgG, IgM, and IgA by DIFFU plate or radial immunodiffussion plate
Radial immunodiffusion (RID) plate (DIFFU plate) containing uniform mono-specific IgG, IgM, and IgA antiserum directed against serum IgG, IgM, and IgA protein in agarose gel layer was used for investigation (Biocientifica S.A., Argentina). The serum samples (5 μL each) were filled on the wells of agarose gel. Wet cotton was placed at the center of the RID plate to avoid agarose dehydration. The plate was tightly closed and incubated at room temperature for 48 h. Radial diffusion of protein out of the well into the surrounding gel led to the formation of a visible precipitation ring by reaction between IgG protein and antiserum. The diameter of the precipitation ring was proportionate to protein concentration and the concentration was determined by the corresponding reference table which was mentioned in the kit.
Determination of immunoglobulin by ELISA
Immunoglobulin (IgE) level was evaluated in the sera using anti-IgE antibody–coated well (Kit-Microwell Total IgE EIA Syntron Bioresearch, California; Cat log No. #2810-96). Absorbance was read at 450 nm using ELISA reader (Lisa Plus Microplate ELISA Reader). Serum IgE was calculated in IU/mL. Each sample was tested in duplicate.
Estimation of copper, manganese, and zinc in blood by inductively coupled plasma-atomic emission spectroscopy
The metal content of the blood samples was analyzed through inductively coupled plasma atomic emission spectroscopy (ICP-AES) using multielement analysis technique. A detector measures the intensity of the emitted light and calculates the concentration of the particular element in the sample. Metal concentration was determined according to the methods of Bazzi et al. and Henk., Each sample was checked after running of separately prepared mix standard in ICP-AES. Metal concentration of copper, manganese, and zinc was calculated in PPM or mg/L.
Data analysis was carried out using SPSS (version 19) and R-3.0.1 programming language with prevalidated programs. Analysis of covariance (ANCOVA) was carried out in all subjects among various study groups independently to adjust possible risk factors such as age, body mass index (BMI) and habitual characteristics (i.e. smoking, tobacco chewing, and alcohol consumption) to assess the true effect of exposure in them. One-way analysis of variance (ANOVA) was then used to calculate statistically significant difference in the overall mean adjusted values of various parameters across the study groups. This was followed by Tukey's post hoc comparison analysis. P value <0.05 was considered to be statistically significant for all the analyses. Linear regression model was also undertaken using logistic regression analysis. The resulting model fitness was evaluated by referring to Hosmer–Lemeshow test. Odds ratios (ORs) were obtained as a measure of effect of varying levels of copper, manganese, and zinc concentrations as risk factors. The normal and abnormal ranges of each parameter were considered as two categories (dichotomous outcomes) dependent on the three metals, which were treated as independent predictors. OR more than 1.1 for corresponding metals was considered as risk factor(s). P value <0.05 was considered statistically significant for all the analyses.
| Results|| |
Analysis of immunoglobulin parameters
Assessment of immunological parameters among the study groups was made post obviation of the influence of confounders such as age, BMI, and several habitual characteristics using ANCOVA. Post adjustment, ANOVA was performed to assess significant differences, if any, in the above parameters across the study groups which has been presented [Table 1]. It is apparent from the table that both these experimental groups exhibited significant decrease (P < 0.0001) in the content of serum IgM when compared with normal control. On the other hand, contents of serum IgG, IgA, and IgE were found to be significantly elevated (P < 0.0001) in both the study groups when compared with normal control as substantiated by ANOVA.
On examination of Tukey's post hoc comparisons, it was distinctly clear that contents of serum IgG and IgA were significantly elevated (P < 0.0001) in miner when compared with office employees and normal control. Contrarily, the content of serum IgM was found to be significantly diminished in both the groups under study when compared with normal control (P < 0.0001), though the extent of decrease was more extensive in miner when compared with office employees. It may be noticed that serum IgE was found to be elevated more significantly only in miner when compared with normal control.
Metal concentration in blood
Examination of blood copper and other associated metals in chronically exposed mine workers and office employees after obviation with covariates revealed statistically significant alterations in the concentration of blood copper and manganese in miner and office employees when compared with normal control as substantiated through one-way ANOVA [Table 2].
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Whereas chronically exposed mine workers and office employees exhibited significantly higher contents of copper (P < 0.0001) when compared with normal control when Tukey's post hoc comparisons were made; the content of manganese was found to be significantly altered only in miner (P < 0.05) when compared with either control groups (normal and office employees). On the other hand, there was insignificant alteration in the level of blood zinc (P > 0.05) across the study groups.
Correlation analysis of metals with immunoglobulin parameters
Pearson's correlation coefficients of copper level showed a significant and positive correlation (P < 0.0001; r = 0.39 with IgE [Figure 1]; P < 0.001; r = 0.28 with IgG [Figure 2]; and P < 0.01; r = 0.21 with IgA [Figure 3], respectively). Whereas a significant but negative correlation (P < 0.0001; r = −0.39) was obtained with IgM [Figure 4]. On the other hand, the above coefficient between Mn and IgG showed some statistical significance (P < 0.05) and a positive correlation (r = 0.18) [Figure 5]. Whereas Zn showed a significant (P < 0.05) but negative correlation (r = −0.20) with IgM [Figure 6]. The summarized form is represented in [Table 3].
|Table 3: Pearson's correlation between metals and immunoglobulin parameters|
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Delineation of risk with immunoglobulin parameters
The effect of change in metal concentrations on immunoglobulin parameters when evaluated through logistic regression analysis showed that a unit increase in Cu concentration enhanced the likelihood of change in IgE level from normal to abnormal by 2.77 times [95% confidence interval (CI): 1.36, 5.63] with a P value <0.05. On the other hand, increase in Cu level decreased the odds in favor of abnormal IgM 0.59 times [95% CI: 0.35, 0.98] with a P value of 0.04 (P < 0.05). Other immunoglobulins were not found to be affected by changing Cu levels. None of these parameters was found to be significantly affected by the changing levels of Mn. Although increase in Zn levels afforded a decrease in the odds in favoring abnormal high IgE levels 0.74 time [95% CI: 0.58, 0.93], no other immnunoglobulins obtained abnormality with changing levels of zinc [Table 4].
Evaluation of the effects of long-term exposure to copper dust (Miner-II) after adjustment for other predictors such as age, BMI, smoking, tobacco chewing, and alcoholism on systemic level of copper and other accompanying metals such as zinc and manganese revealed that there were significant enhancement in the contents of copper in the blood of miner and office employees when compared with normal control. Similar evidence of high levels of blood copper in people from residential area in proximity to copper smelting complex with the possibility of health hazard risk is available. Interestingly, manganese, one of the associated metals in copper mining ore, was found to be raised in the blood of chronically copper dust–exposed miners which is a new finding not hitherto reported. Incidentally, employees residing in the vicinity of mine did not register such a raise in blood manganese.
The present observation of elevated contents of IgG, IgA, IgE, and decreased IgM in the serum of chronically exposed pit workers suggests a compromised immune system in them. This finding is in total congruence with the reported immunological profile in arsenic-exposed human. It may be, however, recalled that almost similar elevations in immunological parameters such as IgG, IgA, and IgE were observed in manganese, chromate, mercury, and lead exposed in human with the exception of IgM which was not found to be varied.,,, It is difficult to comprehend the factors responsible for anomalous immunological alterations observed here, since no genomic evaluation was performed. However, repercussion to such immunological hypersensitivity on health morbidity could be envisaged.
Association of oxidative stress with excess immunogenicity is well-recognized.,,, Hence, it is no wonder that the observed hypersensitivity in pit workers chronically exposed to copper dust would bring about deleterious redox expression as evinced earlier (communicated). It may be recalled that Ercal et al. indeed found enhanced level of IgE with association of exaggerated MDA linked to in lead-exposed workers., Association of type 1 and 2 diabetes, asthma, arthritis, respiratory disorder, and inflammatory diseases with certain alteration in various immunoglobulins has been recognized.,,,,,, In addition, a significant positive correlation between IgE and MDA was observed in patients with asthma. Elevated levels of serum immunoglobins IgA and IgG suggest altered immune response contributing to the pathogenesis of periodontitis in patients with poorly controlled diabetes. IgA levels have been recorded to be high in autoimmune condition. Thus, our observation of general hyperimmunoglobulinemia with diminished IgM in chronically exposed pit worker of a copper mine clearly implicates to probable morbidities such as asthma, allergic condition, diabetes, cardiovascular, respiratory problems, and liver and kidney dysfunction in them.
Analysis of Pearson's correlation of several serum immunoglobulin such as IgG, IgE, and IgA was found to be positively correlated with copper but negatively so with IgM. Further analysis demonstrated that IgG was found to be positively correlated with manganese, while zinc was found to be negatively correlated with IgM However, odd ratio analysis pointed out that out of these parameters only IgM and IgE were affected by increase in copper levels, whereas IgE was found to be affected by increase in zinc levels. Hence, these observations stipulate that affected miners are prone to morbidity such as type 2 diabetes and respiratory discomfort (asthma and hypersensitivity) since imbalance in both IgM and IgE is known to be associated with such morbidity.,,
Although office employees who reside in the vicinity of mine were not directly affected by copper dust exposure but indirectly exposed to metal through water and soil (dietary intake), they did not suffer from immune hypersensitivity with the exception of IgM which was significantly decreased showing proneness to asthmatic conditions.
Hartwell TD, Handy RW, Harris BS, Williams SR, Gehlbach SH. Heavy metal exposure in populations living around zinc and copper smelters. Arch Environ Health 1983;38:284-95.
Jung MC, Thornton I. Environmental contamination and seasonal variation of metals in soils, plants and waters in the paddy fields around a Pb-Zn mine in Korea. Sci Total Environ 1997;198:105-21.
McLaughlin JK, Chen JQ, Dosemeci M, Chen RA, Rexing SH, Wu Z, et al.
Anested case-control study of lung cancer among silica exposed workers in China. Br J Ind Med 1992;49:167-71.
Agency for Toxic Substances and Disease Registry. Toxicological Profile for Copper. United States Public Health Service; 1990.
Gerhardsson L, Englyst V, Lundström NG, Sandberg S, Nordberg G. Cadmium, copper and zinc in tissues of deceased copper smelter workers. J Trace Elem Med Biol 2002;16:261-6.
Tumane RG, Pingle SK, Jawade AA, Nath NN. An overview of caspase: Apoptotic protein for silicosis. Indian J Occup Environ Med 2010;14:31-8.
] [Full text]
Tumane R, Nath N, Khan A. Assessment of blood biochemistry and hematological parameters in copper pit workers and associated employees. J Appl Environ Biol Sci 2017;7:99-118.
Mishra KP, Chauhan UK, Naik S. Effect of lead exposure on serum immunoglobulins and reactive nitrogen and oxygen intermediate. Hum Exp Toxicol 2006;25:661-5.
Ercal N, Neal R, Treeratphan P, Lutz PM, Hammond TC, Dennery PA, et al.
Arole for oxidative stress in suppressing serum immunoglobulin levels in lead-exposed fisher 344 rats. Arch Environ Contam Toxicol 2000;39:251-6.
Bazzi A, Nriagu JO, Linder AM. Determination of toxic and essential elements in children's blood with inductively coupled plasma-mass spectrometry. J Environ Monit 2008;10:1226-32.
Henk JV. Determination of Elements by ICP-AES and ICP-MS. Bilthoven, The Netherlands: National Institute of Public Health and the Environment (RIVM); 2003.
Raikwar MK, Kumar P, Singh M, Singh A. Toxic effect of heavy metals in livestock health. Veterinary World 2008;1:28-30.
Hutton M. Human health concerns of lead, mercury, cadmium and arsenic. Lead, Mercury, Cadmium and Arsenic in the Environment. Edited by T. C. Hutchinson and K. M. Meema @ SCOPE. John Wiley & Sons Ltd.; 1987.
Raja R, Namburu S. Impact of heavy metals on environmental pollution. J Chem Pharm Sci 2014;3:175-81.
de Burbure C, Buchet JP, Leroyer A, Nisse C, Haguenoer JM, Mutti A, et al.
Renal and neurologic effects of cadmium, lead, mercury, and arsenic in children: Evidence of early effects and multiple interactions at environmental exposure levels. Environ Health Perspect 2006;114:584-90.
Fraga CG. Relevance, essentiality and toxicity of trace elements in human health. Mol Aspects Med 2005;26:235-44.
Treagan L. Metals and the immune response. A review. Res Commun Chem Pathol Pharmacol 1975;12:189-220.
Wilde KD. The Effect of Chronic Exposure to Low Levels of Heavy Metals in Immune Mechanism in the Guinea Pig. Ph. D thesis, Health Science, Immunolgoy, University of Maryland, Baltimore; 1976.
Boscolo P, Di Gioacchino M, Bavazzano P, White M, Sabbioni E. Effects of chromium on lymphocyte subsets and immunoglobulins from normal population and exposed workers. Life Sci 1997;60:1319-25.
Hedin N. Metal Ion Interactions with Immunoglobulin G (IgG). Preliminary Studies with Electron Paramagnetic Resonance (EPR) Spectroscopy and Ultrafiltration. South Dakota School of Mines and Technology Rap-Ty, South Dakota; 1979.
Sakhaee E, Behzadi MJ, Ellieh S. Subclinical copper poisoning in asymptomatic people in residential area near copper smelting complex. Asian Pac J Trop Dis 2012;2:475-7.
Islam LN, Nabi AH, Rahman MM, Zahid MS. Association of respiratory complications and elevated serum immunoglobulins with drinking water arsenic toxicity in human. J Environ Sci Health A Tox Hazard Subst Environ Eng 2007;42:1807-14.
Khadiga SI, El-Attar MS, El-Din H, El-Anwar A, El-Mishad AM, Abd-Alla HM. Some health hazards of manganese exposure among Egyptian workers. Central Eur J Occup Environ Med 2001;7:228-36.
Qian Q, Li P, Wang T, Zhang J, Yu S, Chen T, et al.
Alteration of Th1/Th2/Th17 cytokine profile and humoral immune responses associated with chromate exposure. Occup Environ Med 2013;70:697-702.
Queiroz ML, Perlingeiro RC, Dantas DC, Bizzacchi JM, De Capitani EM. Immunoglobulin levels in workers exposed to inorganic mercury. Pharmacol Toxicol 1994;74:72-5.
Thanoon IA. Oxidative stress and immunoglobulin levels in patients with Hodgkin's lymphoma. Med J Basrah Univ 2007;25:23-7.
Da S. Oxidative stress and immunoglobulin levels in workers exposed to cotton dust. Egypt J Occup Med 2012;36:107-21.
Lutz PM, Wilson TJ, Ireland J, Jones AL, Gorman JS, Gale NL, et al.
Elevated immunoglobulin E (IgE) levels in children with exposure to environmental lead. Toxicology 1999;134:63-78.
Zhian M, Dezayee I, Alnakshabandi AQ. Assessment of Some immunological parameters in respect to glycemic control in type 1 and 2 diabetes mellitus: Comparative study. Res J Med Sci 2011;5:119-21.
Awartani F. Serum immunoglobulin levels in type 2 diabetes patients with chronic periodontitis. J Contemp Dent Pract 2010;11:001-8.
Anupama N, Sharma MV, Nagaraja HS, Bhat MR. The serum immunoglobuline level reflects the severity of bronchial asthma. J Physiol Sci 2005;18:35-40.
Aho K, Heliövaara M, Knekt P, Reunanen A, Aromaa A, Leino A, et al.
Serum immunoglobulins and the risk of rheumatoid arthritis. Ann Rheum Dis 1997;56:351-6.
Petty RE, Palmer NR, Cassidy JT, Tubergen DG, Sullivan DB. The association of autoimmune diseases and anti-IgA antibodies in patients with selective IgA deficiency. Clin Exp Immunol 1979;37:83-8.
Arkwright PD, Abinun M, Cant AJ. Autoimmunity in human primary immunodeficiency diseases. Blood 2002;99:2694-702.
Zinah A, Mufeed E, Gaylany A, Hamid H. The relationship of immunoglobulin-e and oxidative stress in Iraqi patients with asthma. Adv Life Sci Technol 2014;18:93-5.
Arnason JE, Campigotto F, Neuberg D, Bussel JB. Abnormalities in IgA and IgM are associated with treatment-resistant ITP. Blood 2012;119:5016-20.
Sandeep T, Roopakala MS, Silvia CR, Chandrashekara S, Rao M. Evaluation of serum immunoglobulin E levels in bronchial asthma. Lung India 2010;27:138-40.
] [Full text]
Deo SS, Mistry KJ, Kakade AM, Niphadka PV, Relationship of total IgE, specific IgE, skin test reactivity and eosinophils in Indian patients with allergy. J Indian Acad Clin Med 2010;11:265-71.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]