Risk Assessment in Mining-Based Industrial Workers by Immunological Parameters as Copper Toxicity Markers.

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.

INTRODUCTION

Various ill effects on workers were associated with copper industries as smelting, brassing, and wiring have been reported earlier.[12] Certain studies indicate that workers exposed to copper dust from smelters grow signs of hepatomegaly, digestive disorders, and a range of respiratory distress.[345] Magnitudes of these effects were found to be largely depended on both the level and length of exposure.[6] Deposition of copper in liver, brain, kidney, and lung among dead workers at copper smelters has also been evidenced.[7] Surprisingly, reports are unavailable regarding the toxic effects of this metal in subjects working in open pit especially with regard to the tenure of exposure in such mining operations. Yes, it may be recalled that recently pit workers in a copper mine and office employees residing in the vicinity indeed were found to suffer from health morbidity.[8] A systematic evaluation of their probable association with blood biochemical parameters and hematological parameters have also been presented in workers at actual mining area.[9] Furthermore, they were found to show certain redox anamolies (communicated). Since alteration in immunity profile has been linked to oxidative stress,[1011] attempts were made here to examine the probable anomalies if any, in the immunoglobulin profile, and thus humoral immunity among the miners chronically exposed to copper dust as well as office employees residing in the vicinity of mine. Hence, a comprehensive examination in the contents of serum IgG, IgM, IgA, and IgE of workers in a copper mine has been presented here.

Besides, analysis of risk associated with exposure of copper dust in chronically exposed miner and office employee's residing in the vicinity of mine was not assessed earlier. Delineation of the effect of associated metals from copper dust as a risk factor (s) therefore needs to be carefully evaluated. This study was undertaken to obtain a cogent view regarding the variations, if any, in various immunological parameters among the study groups as associated with metal covariates present in blood by using appropriate statistical tools.

MATERIALS AND METHODS

Selection of subjects

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.[9] 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.[8]

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.[1213] 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.

Statistical analysis

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.

Table 1

Immunoglobulin parameters in all study groups after adjustment

Characteristics	Groups			P*	Pair-wise comparisons‡
	Controls (n=30)	Office employees (n=47)	Miner-II (n=71)		Control versus office employees	Control versus Miner-II	Office employees versus Miner-II
Ig parameters
IgG (mg/dL)	524.55+320.51	568.4+417.18	1172.82+493.23	<0.0001***	0.9045	<0.0001***	<0.0001***
IgM (mg/dL)	247.17+123.48	162.16+99.32	83.87+51.84	<0.0001***	0.0001***	<0.0001***	<0.0001***
IgA (mg/dL)	198.09+109.52	232.94+137.52	394.27+149.98	<0.0001***	0.5312	<0.0001***	<0.0001***
IgE (IU/mL)	235.12+136.37	356.5+226.91	393.67+259.09	0.0073	0.0636	0.0051*	0.6644

Ig: Immunoglobulin; BMI: Body mass index; ANOVA: Analysis of variance. †Adjusted for age, bmi, smoking, tobacco and alcohol; *P-value obtained using one way ANOVA; ‡Tukey’s post hoc comparisons Bold numbers indicate statistical significance. *denotes P value less than < 0.05 as considered statistically significant . **denotes P value less than < 0.001as considered highly significant . *** denotes P value less than < 0.0001as considered extremely significant.

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].

Table 2

Blood metal analysis in various study groups post adjustment with confounder

Biochemical parameters	Groups			P*	Pair-wise comparisons‡
	Controls (n=30)	Office workers (n=47)	Miners-II (n=71)		Control versus office workers	Control versus Miner-II	Office workers versus Miner-II
Cu (mg/L)	0.66+0.34	2.14+0.84	2.22+0.79	0.0001***	0.0001***	0.0001***	0.8206
Mn (mg/L)	1.09+0.65	1.89+1.35	2.09+1.85	0.0118*	0.0674	0.0087*	0.7686
Zn (mg/L)	7.31+1.19	8.44+2.46	8.1+3.01	0.1699	0.1482	0.3385	0.7618

BMI: Body mass index; Cu: Copper; Mn: Manganese; Zn: Zinc; ANOVA: Analysis of variance. †Adjusted for age, BMI, smoking, tobacco, and alcohol; *P-value obtained using one-way ANOVA; ‡Tukey’s post hoc comparisons; values in bold indicate statistical significance, *P<0.05 considered as statistically significant, **P<0.001 considered as highly significant, ***P<0.0001 considered as extremely significant

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.

Figure 1

Positive correlation between IgE and copper

Figure 2

Positive correlation between IgG and copper

Figure 3

Positive correlation between IgA and copper

Figure 4

Negative correlation between IgM and copper

Figure 5

Positive correlation between IgG and manganese

Figure 6

Negative correlation between IgM and zinc

Table 3

Pearson’s correlation between metals and immunoglobulin parameters

Ig parameters	Correlation coefficient (P)Metals
	Cu	Mn	Zn
IgG (mg/dL)	0.28 (0.001)**	0.18 (0.03)*	−0.004 (0.96)
IgM (mg/dL)	−0.39 (<0.0001)***	−0.10 (0.24)	−0.20 (0.01)*
IgA (mg/dl)	0.21 (0.01)*	0.09 (0.27)	−0.03 (0.68)
IgE (IU/mL)	0.39 (<0.0001)***	0.14 (0.08)	0.01 (0.90)

Ig: Immunoglobulin; Cu: Copper; Mn: Manganese; Zn: zinc. *P value less than < 0.05 as considered statistically significant, **P value less than < 0.001as considered highly significant, ***P value less than < 0.0001as considered extremely significant

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].

Table 4

Odds ratio for different immunological parameters after adjusting for covariates using logistic regression*

Ig parameters	OR (95% CI) (P)			Classification accuracy (%)	HL test
	Cu	Mn	Zn
IgG	0.66 (0.42-1.04) 0.07	1.09 (0.85-1.38) 0.5	1.06 (0.91-1.24) 0.42	62.16	0.4433
IgM	0.59 (0.35-0.98) 0.04	0.92 (0.70-1.21) 0.57	0.95 (0.79-1.14) 0.58	70.27	0.6877
IgA	1.45 (0.82-2.57) 0.2	0.93 (0.69-1.23) 0.6	1.00 (0.82-1.22) 0.99	81.08	0.1729
IgE	2.77 (1.36-5.63) 0.004	1.39 (0.81-2.37) 0.23	0.74 (0.58-0.93) 0.01	85.81	0.7998

*Cut-offs of respective parameters have been used to determine the odds ratio. HL: Hosmer-Lemeshow test, Cu: Copper, Mn: Manganese, Zn: Zinc, OR: Odds ratio, CI: Confidence interval, Ig: Immunoglobulin. *denotes P value less than < 0.05 as considered statistically significant . **denotes P value less than < 0.001as considered highly significant.

DISCUSSION

Occupational or environmental exposure to heavy metal is believed to affect human health adversely even at moderate levels of exposure.[14151617] Some metals and their complexes pose a major environmental and industrial problem due to their reported toxic effects.[18] Occupational exposure to certain metals, such as Co, Ni, Zn, Cr, and Be, has been linked to clinical hypersensitivity and especially contact hypersensitivity, the most common occupational disease.[19202122] Although nickel chloride showed a consistently suppressive effect on the interferon system (component of the immune system), salts of iron, chromium, cadmium, thorium, or lead did not.[23] This is probably the first ever attempt that has been made to screen the immunological status of copper pit workers.

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.[24] 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.[25] 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.[26272811] 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.[10112930] 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.[1131] Association of type 1 and 2 diabetes, asthma, arthritis, respiratory disorder, and inflammatory diseases with certain alteration in various immunoglobulins has been recognized.[25323334353637] In addition, a significant positive correlation between IgE and MDA was observed in patients with asthma.[38] Elevated levels of serum immunoglobins IgA and IgG suggest altered immune response contributing to the pathogenesis of periodontitis in patients with poorly controlled diabetes.[33] IgA levels have been recorded to be high in autoimmune condition.[39] 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.[8]

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[33] and respiratory discomfort (asthma and hypersensitivity) since imbalance in both IgM and IgE is known to be associated with such morbidity.[344041]

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.

CONCLUSION

Serum IgG and IgA were found to be elevated in chronically exposed pit workers when compared with both office employees and normal control. However, the content of corroborate serum IgM was recorded to be decreased in both the groups under study when compared with normal control. Serum IgE was found to be elevated significantly in only chronically exposed pit workers when compared with normal control subjects. These observations of elevated contents of IgG, IgA, and IgE and decrease in IgM in the serum of chronically exposed pit workers suggest a compromised immune system in them.

Several serum immunoglobulins such as IgG, IgE, and IgA were found to be positively correlated with copper but negatively with IgM. OR analysis substantiates that out of these parameters only IgM and IgE were affected by increase in copper levels. IgE was also found to be affected by increase in zinc levels. Thus, it could be inferred from these findings that 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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.