Association of Musculoskeletal Disorders and Inflammation Markers in Workers Exposed to Lead (Pb) from Pb-battery Manufacturing plant.

BACKGROUND: Lead (Pb) deposits in the skeletal system on chronic exposure and releases to circulation over a period. The musculoskeletal disorders (MSDs) are associated with enhanced expression of inflammation. The combination of Pb-exposure and MSDs induced inflammation was not attempted.
OBJECTIVE: This study was conducted to examine the association between MSDs and inflammatory markers in workers exposed to Pb from Pb-battery plant.
MATERIAL AND METHODS: In a case-control study design, the study enrolled 176 male Pb-exposed workers as study subjects and 80 healthy workers with no occupational exposure to Pb as control subjects. The Nordic musculoskeletal questionnaire was used to assess the MSDs. From the blood sample, blood lead level (BLL) and High Sensitivity C-reactive protein (Hs-CRP) were estimated as markers of Pb-exposure and Inflammatory marker respectively. The BLL was estimated by flame atomic absorption spectrometric method and the Hs-CRP by using a diagnostic kit method.
RESULTS: Significantly high proportions of MSDs were noted in study subjects as compared to controls. The MSDs identified in the study subjects were at low back (33%) followed by knee (26%), shoulders (16%), neck (14%), ankle/foot (11%), wrist/hand (10%), elbows (8%), upper back (7%), and hips/thighs (5%). The significant association between Pb-exposure and MSDs among study subjects was mainly noted in low back and ankle/foot. Also, significantly high serum Hs-CRP levels were noted among study subjects with ankle/foot MSDs.
CONCLUSION: Pb-exposure and inflammatory markers were significantly associated with lower limbs of MSDs. Copyright:

INTRODUCTION

Manufacturing of Lead (Pb) batteries is involved in the preparation of lead oxide, grid casting, pasting, plate cutting, formation, charging, and assembly. The chemicals used in these processes are hazardous in nature; they comprise of lead oxide (PbO2), spongy lead (Pb), and sulfuric acid (H2 SO4). Pb can enter the bodies of workers through inhalation and ingestion. Inhalation is the primary route of exposure in Pb-related occupations.[1] In the body, Pb accumulates in erythrocytes, soft tissues (brain, kidney, and bone marrow), and mineralized tissue (bone, teeth). Literature states that workers exposed to Pb form Pb-battery manufacturing plant have reported the disturbances in epigenetic status,[2] oxidative DNA damage,[3] altered reticulocyte count,[4] oxidative stress,[5] biogenic amino acids with neurobehavioral changes,[6] gene polymorphisms of ALAD, glutathione peroxidase (GPx), paraoxonase and metallothionein-4 activities [789], and calcium metabolism with bone mineral density.[10] Pb also affects several body systems, which include blood pressure,[11] ocular changes,[12] coagulation,[13] osteoporosis risk [14] poor dental health,[15] hearing loss [16] cardiovascular,[17] neuropathy,[18] renal,[19] hepatic [20], and reproductive [21] abnormalities.

Musculoskeletal disorders (MSDs) are work-related ailments. It affects multiple body parts at the back, neck, shoulders, and upper and lower limbs. Physical and organizational hazards are the significant contributors of work-related MSDs. The causes of MSDs include the handling of loads, repetitive or forceful movements, awkward and static postures, vibration, poor lighting or cold working environments, and prolonged sitting or standing in the same position. Musculoskeletal components such as motor skills, bones growth and development, dentition, fracture healing, bone density, and joint maintains are susceptible to Pb.[22] Kuruvilla et al.[23] reported the prevalence of backache, muscular exhaustibility, myalgia, and paresthesia in workers from printing and lead-battery plant. It was reported that the MSD morbidities among Pb-battery manufacturing workers were significantly associated with odds of blood lead levels (BLLs).[24] A recent study reported high prevalence of pain in limbs, weakness of limbs, and numbness of limbs among these Pb-workers.[25] Also, in severe Pb-poisoning generalized weakness predominates in proximal limbs.[26] It is well documented in the literature that the Pb-exposure enhances the expression of inflammation.[2728] Pb-induced inflammation causes the disorders of various organs such as respiratory, neurologic, digestive, cardiovascular, and urinary diseases [29]; however, there is limited literature on musculoskeletal systems. It has been observed that the farmers with MSD had altered plasma levels of protein biomarkers compared to the referents, indicating that farmers with MSD had more systemic inflammation.[30] Healthy females with work-related neck/shoulder pain showed higher serum concentrations of CRP than controls, and the levels of CRP were correlated to pain intensity.[31] The review of literature suggests that the Pb-exposure and the presence of MSDs are associated with inflammation. However, there is limited literature about the association between the Pb-exposure and MSDs along with inflammatory biomarkers. This study is intended to find out the association between MSDs of different parts of body regions and inflammatory markers in workers exposed to Pb from Pb-battery manufacturing plant.

MATERIALS AND METHODS

The study used case-control study design. The workers engaged in Pb-battery manufacturing plant and exposed to Pb with >2 years were considered as study subjects.

The study involved a total of 256 male subjects, including both study subjects and controls. The study group consisted of 176 workers, who were working in Pb-battery manufacturing plant located in Tamilnadu, India. Eighty office workers with no occupational exposure to Pb were considered as a control group. The control subjects were matched for age and the socioeconomic status of study group. The institutional ethical committee approved the study. Informed written consent was obtained from each of the subject before participation in the study. Demographic details, work history, and habits of the subjects were obtained through a validated questionnaire.

Blood sample collection

From each subject 4 ml (2 ml in heparin tubes + 2 ml in plain tubes) of whole blood was collected. 2 ml of heparinised blood was used for the determination of BLL. 2 ml of whole blood sample was collected in plain tubes, centrifuged at 4000 RPM for 10 min at 4°C for the separation of serum and red blood cells. The serum samples were used for the assessment of inflammatory markers.

Blood lead

The BLLs were measured as mentioned previously in Barman et al.[13] Briefly, in this method, 2 ml of whole blood sample was digested by a microwave digestion system (ETHOS-D, Italy) with 2 ml of nitric acid (HNO3) and 0.2 ml of hydrogen peroxide (H2O2). The digested samples were made up to 5 ml using distilled water and centrifuged. The BLL was measured by an atomic absorption spectrophotometer (GBC Avanta, Australia).

Inflammatory and anthropometry

Inflammatory and anthropometry assessment was performed using the guidelines of Soeters et al.[32] (i) Anthropometry (body weight, height, and body mass index) and (ii) measurement of inflammatory activity (albumin, hemoglobin, and Hs-C-reactive protein).

Body mass index

BMI was calculated by using subject's weight (Kg) by squared height (m) and expressed as Kg/m2.

Serum albumin

The serum albumin concentration was measured by using bromocresol green method of Doumas et al.[33] In this method, albumin in a buffered solution reacts with the anionic bromocresol green dye and gives a green color that was measured at 628 nm. The intensity of green color was directly proportional to the concentration of albumin present in the sample. The results were expressed as g/dL of sample.

Hemoglobin

The hemoglobin content in samples was measured by using the method developed by Drabkin and Austin.[34] In this method, 20 μL of blood is mixed with 5 mL of Drabkin's solution that contains ferricyanide and cyanide. The ferricyanide oxidizes hemoglobin into methemoglobin. Methemoglobin then unites with the cyanide to form cyanmethemoglobin and which produces a color that is measured at 540 nm using a colorimeter (Elico, CL-223). The level of hemoglobin is expressed as g/dL.

Serum high sensitivity C - reactive protein (Hs-CRP)

The concentration of Hs-CRP in serum was determined using latex turbidimetric immunoassay. Latex particles coated with purified anti-CRP, when allowed to react with samples containing CRP agglutinate causing a change in absorbance. The change of absorbance depends on the concentration of CRP and is determined by comparing it with the known concentration of the calibrator. The concentration of CRP in the samples was expressed as mg/L. The detection limit of the method is 1 mg/L and measuring range is 1−160 mg/L.

Musculoskeletal disorders (MSDs)

The Nordic musculoskeletal questionnaire (NMQ) was used to identify the MSDs among study and control subjects.[35] This questionnaire assesses the frequency of symptoms in all parts of the body. Choi et al.[36] validated the questionnaire of NMQ for assessing the MSDs. Validated data of NMQ were found to have the sensitivity of 73.9%, specificity of 68%, positive predict value of 72.6%, negative predictive value of 69.5%, and kappa coefficient is 0.42.

Statistical analysis

Data analysis was performed using SPSS version 16.0 (SPSS). The data were evaluated using the obtained mean and SD and compared with Student's t-test between study subjects and controls. The Chi-squared test was used to compare the MSDs distribution among study and control group. Spearmen correlation coefficient (r) test was used to find out the association between BLLs and inflammatory markers. The differences of BLLs and inflammatory in workers with and without MSDs were computed by using nonparametric Mann-Whitney U test. P value of < 0.05 was considered statistically significant.

RESULTS

The demographic details of study and control subjects were presented in Table 1. The study parameters such as age, blood pressure (SBP and DBP), frequency distribution of alcohol consumption, and smoking habits did not differ between study and controls. Significantly elevated BLLs were noted in study subjects as compared to controls. The frequency distribution of elevated BLLs among study subjects were reported as per the set guidelines of American Conference of Governmental Industrial Hygienist-Biological Exposure Index.

Table 1

Demographic detail of study and control subjects

Variables	Study (176)	Controls (80)	Probability
Age (years)	36.6±4	37.4±10	0.473
Experience (years)	13.3±3.3	0.00	-
BMI (Kg/m2)	25.7±3.0	25.3±3.3	0.360
DBP (mmHg)	77.7±10.7	74.8±12.7	0.083
SBP (mmHg)	127.8±14.6	127.5±19.1	0.889
Alcohol consumption [n (%)]	95 (54)	39 (49)	0.436
Smoking [n (%)]	38 (22)	19 (24)	0.700
Blood lead levels (µg/dL) [n (%)]
≤ 30 µg/dL	84 (48)	70 (88)	0.000
>30 µg/dL	92 (52) *	10 (12)

*P<0.005

The frequency of MSDs in the study and control subjects at different body regions were tabulated in Table 2. Significantly high proportion of MSDs was observed in study subjects as compared to controls. In study subjects, the highest proportion of MSD condition was identified at lower back ache (33%), followed by the knee (26%), shoulders (16%), neck (14%), ankle/feet (11%), wrist/hand (10%), elbows (8%), upper back (7%), and hips/thighs (5%).

Table 2

Musculoskeletal disorders in study and control subjects according to body regions

Body regions	Study (176)	Controls (80)	P
Neck [n (%)]	24 (14)	03 (3.7)	0.015*
Shoulders [n (%)]	28 (16)	04 (5.0)	0.014*
Elbows [n (%)]	14 (8)	00 (0.0)	0.006*
Wrist/Hands [n (%)]	17 (10)	01 (1.3)	0.015*
Upper back [n (%)]	12 (7)	00 (0.0)	0.021*
Lower back [n (%)]	58 (33)	07 (8.7)	0.0008
Hips/Thighs [n (%)]	08 (5)	01 (1.3)	0.281
Knees [n (%)]	46 (26)	12 (15)	0.054*
Ankle/feet [n (%)]	20 (11)	00 (0.0)	0.001*

*P<0.05

The means and standard deviation values of BLLs and inflammatory biomarkers in Pb-exposed workers with or without MSDs were presented in Table 3. Significantly higher BLLs were observed among subjects with MSDs at upper back, lower back, and ankle/feet as compared to without MSDs. Higher serum Hs-CRP was observed among subjects with ankle/feet MSD as compared to subjects without MSD.

Table 3

Blood lead levels and inflammatory markers in subjects with and without MSD

MSD Body region		BLLs (µg/dL)	Hb%	Albumin (g/dL)	Hs-CRP (mg/L)
Neck	Yes (27)	31.8±15	14.4±1.1	3.8±0.3	4.4±4.6
	No (229)	27.5±13	14.3±1.2	4.1±0.5	3.3±3.0
Shoulders	Yes (32)	27.4±12	14.3±1.0	4.2±0.5	3.5±2.9
	No (224)	28.1±14	14.3±1.2	4.0±0.5	3.4±3.2
Elbows	Yes (14)	31.5±12	14.5±1.3	4.0±0.3	4.0±3.1
	No (242)	28.0±13	14.3±1.2	4.1±0.5	3.4±3.2
Wrist/hands	Yes (18)	31.8±14	14.2±1.1	4.1±0.4	4.2±2.8
	No (238)	28.0±13	14.3±1.2	4.1±0.5	3.4±3.2
Upper back	Yes (12)	36.8±16*	14.6±1.5	4.1±0.5	5.7±6.5
	No (244)	28.0±13	14.3±1.2	4.1±0.5	3.3±2.9
Lower back	Yes (65)	33.1±13*	14.4±1.1	4.1±0.6	4.0±3.7
	No (191)	26.2±13	14.2±1.2	4.1±0.4	3.2±3.0
Hips/Thighs	Yes (9)	31.3±13	14.8±2.3	4.4±0.8	4.0±3.7
	No (247)	28.0±13	14.2±1.1	4.1±0.5	3.4±3.2
Knees	Yes (58)	31.0±13	14.2±1.1	4.0±0.5	3.8±3.6
	No (198)	27.2±13	14.3±1.2	4.1±0.5	3.3±3.1
Ankle/feet	Yes (20)	34.6±12*	14.3±1.4	4.1±0.5	5.0±3.2*
	No (236)	27.4±13	14.3±1.2	4.1±0.5	3.3±3.2

*P<0.05 (BLLs=Blood Lead levels, Hb=Hemoglobin, Hs-CRP=high sensitivity C - reactive protein)

The results of spearmen correlation coefficients (r) between BLLS and inflammatory markers among total subjects were presented in Table 4. A significant positive correlation was noticed between BLLs with Hb% and Hs C-reactive protein.

Table 4

Spearmen correlation coefficients (r) between blood lead levels and inflammatory markers

Parameters	BLLs (µg/dL)	Hb%	Albumin (g/dL)	Hs-C-Reactive protein (mg/L)
BLLs (µg/dL)	1.000	-	-	-
Hb%	0.207**	1.000	-	-
Albumin (g/dL)	0.004	0.006	1.000	-
Hs-C-Reactive protein (mg/L)	0.193**	0.040	-0.057	1.000

**Correlation significant at 0.01

DISCUSSION

This study assessed the association between MSDs of different body regions and inflammatory markers in workers exposed to Pb from Pb-battery manufacturing plant. The study observed higher proportions of MSDs in Pb-exposed workers. Significantly higher BLLs were observed among subjects with MSD at upper-back, lower-back, and ankle/feet as compared to without MSDs.

The current literature reports that generalized weakness predominates in proximal limbs in severe Pb-poisoning cases,[26] However, other studies observed backache, muscular exhaustibility, myalgia, and paresthesia in printers and Pb-battery workers.[23] This study also reiterates the high proportion of MSDs among chronic Pb-exposed workers. The MSDs conditions identified in the study subjects were at lower back (33%), followed by knee (26%), shoulders (16%), neck (14%), ankle/feet (11%), wrist/hand (10%), elbows (8%), upper back (7%), and hips/thighs (5%). The literature suggests that the Pb induces an osteoarthritis-like phenotype in articular chondrocytes through the disruption of TGF-β signaling leads disruption normal chondrocyte phenotype.[37] Kalahasthi et al.[24] reported that the odds ratio of BLLs was significantly associated with MSDs in workers from Pb-battery plant. In this study, the Pb-exposure in the form of elevated BLLs was significantly associated with workers who had MSDs at upper and lower back and ankle and foot. Similar observations were also reported in a recent study with high prevalence of pain in limbs, weakness of limbs, and numbness of limbs in Pb-battery workers.[25] The numbness in limbs was significantly associated with BLLs.[38] In this study, in addition to echoing of that finding, we observed that the workers, who had ankle/foot problems, were significantly associated with BLLs.

This study observed higher serum Hs-CRP among subjects with ankle/feet MSD as compared to subjects without MSD. The current literature abridges that the Pb-exposure enhances the expression of inflammation factors and causes the disorders of various organs such as respiratory, neurologic, digestive, cardiovascular, and urinary diseases.[272829] Several evidence of literature from MSD research point out that MSD is associated with higher inflammatory markers, such as farmers with MSD had altered plasma levels of protein biomarkers compared to the referents, suggesting that farmers with MSD had more systemic inflammation.[30] Healthy females with work-related neck/shoulder pain showed higher serum concentrations of CRP than controls, and the levels of CRP were correlated to pain intensity.[31] Thus, most of the studies report that the Pb-exposure and the presence of MSD are associated with inflammation expressions. In this study, we report the association between MSDs, more specifically directed toward lower-limb MSDs and inflammatory markers in workers exposed to Pb from Pb-battery plant. In addition, we also report that higher levels of serum Hs-CRP were noted in subjects with ankle/feet MSD as compared to subjects without MSD. Further, the levels of inflammatory markers were positively associated with BLLs, and a significant association was noted in Hb% and serum Hs-CRP. Pb-exposure and inflammatory marker were significantly associated with lower limbs of MSDs. In conclusion, these findings of higher MSDs in lower limb as well as its significant association with high inflammatory markers suggest that the lower limbs are more influenced by occupational Pb-exposure associated with MSDs. However, more specific studies are warranted to confirm these findings in different occupational Pb-exposed situations.

Financial support and sponsorship

ICMR, New Delhi.

Conflicts of interest

There are no conflicts of interest.