Impact of occupational health hazards on serum markers of bone formation in spray painters of Chennai region in Tamil Nadu.

CONTEXT: The association between spray paint exposure and bone remodeling received little attention despite the high usage of spray paints in automobile industries, steel furniture workshops etc. AIM: The present study was aimed at investigating the level of serum markers of bone formation in spray painters. The spray painting subjects were selected from automobile body repair workshops in Chennai region of TamilNadu which constitutes 30% of India's automobile industry. SETTING AND
DESIGN: All the study subjects, exposed to spray paint were working in a workshop without standard spraying room and did not wore any aerosol removing respirator. The controls were selected from random population irrespective of occupation. Data relevant to the socioeconomic features and personal history was collected using a questionnaire. The current study included 50 spray painters and 25 control subjects of same age group.
MATERIALS AND METHODS: We examined the level of serum calcium, serum phosphorus, serum differentiation markers of bone such as alkaline phosphatase (bone specific) and serum osteocalcin in which these levels were found to be high in serum of spray painters.
CONCLUSION: The current study concludes dysregulation in bone remodeling of spray painters exposed to chronic solvents and paint pigments.

INTRODUCTION

Chemicals in spray paints like Lead, mercury, cadmium, isocyanates (toluene diisocyanate, diphenyl methane diisocyanate, hexamethylene diisocyanate, biuret modified), xylene, benzene, ethylbenzene and methylbenzene (toluene) have been reported to be toxic to human health.[1] Various reports on the toxicity of chemicals in paints to humans and mice models have been published.[2-4] Moreover, the effects of such chemicals on bone have also been demonstrated. But effect of spray paints as such was not studied on bone remodeling. Bone mineralization/resorption has been reported to be affected by lead, which is an ingredient of paints.[5] Lead significantly reduces the bone mineral density, mineral/matrix ratio in cortical bone.[6] Chromate (VI) released from three type of paint particles (solvent borne epoxy, water borne epoxy and polyurethane) accelerated bone resorption activity with decreased bone formation in suckling rats by inducing oxidative stress.[7] Alkylphenol ethoxylate, Formaldehye, phthalates, styrene are the major endocrine disrupting the chemicals present in paints.[89] Toluene exposure results in oxidative stress and induces apoptosis.[10]

These chemicals in spray paints appear to be either endocrine disruptors or a pro-oxidant. In such case, the spray paint chemicals being an endocrine disrupting pollutant may show anti-estrogenic activity[11] resulting in reduced bone mineral density. Butyl benzyl phthalate suppresses the cell proliferation of osteoblast by P2X receptor binding.[12] Alkyl phenol seems to influence bone by non-estrogenic effects.[13] Though many studies on these chemicals were reported on animal and in vitro studies, their effect on human as occupational hazard is very limited. Since many of these compounds were involved in affecting bone remodeling and turnover, we studied the impact of the collective exposure of these chemicals to spray painters in present study using serum differentiation markers.

MATERIALS AND METHODS

Criteria for selecting painters

Painters (n = 50) were selected based on the number of years of handling spray paint within the age group 25-35 years. A minimum of 5 years of exposure was set as criteria to include them in study. Controls (n = 25) were based on criteria such as no exposure to organic solvents, absence of osteoporosis, absence of family history of musculoskeletal disorder. The painters were informed in detail about the research study and consent was obtained which was in conformity to Institute ethical committee's requirements. The health condition was evaluated using a questionnaire. The questionnaire contains details under personal history and occupational history. Occupational history includes years of experience, hours of exposure per day, type of preventive measures if any, type of paint and its chemical constituents.

Sample collection

Peripheral venous blood from brachial vein was collected from spray painters working at various car companies, automobile workshops which were dealing with spray painting around Chennai city. The samples were transferred to serum tubes for analysis.

Serum bone specific alkaline phosphatase

Bone specific Alkaline Phosphatase was measured using Access ostase assay (Hybritech Inc, San Diego, US) which is a one-step immunoenzymetric method in which the bone specific serum ALP or skeletal ALP were allowed to react with biotin labeled bone ALP specific monoclonal antibody. After incubating with enzyme substrate, the turnover was measured calorimetrically at the absorbance of 405 nm.

Serum phosphorus

The Molybdate UV method was adopted to measure serum phosphorous level using phosphorus kit (CREST Biosystems, India). Phosphate ions forms phosphomolybdate complex by reacting with ammonium molybdate in acidic medium. Intensity of complex is proportional to the amount of inorganic phosphorus. This complex was measured at the absorbance at 340 nm.

Serum calcium

The OCPC (o-cresolphthalein complexone) method was adopted to measure serum calcim level using calcium kit (CREST Biosystems, India). Calcium forms a purple colored complex by coupling with o-cresolphthalein complexone in alkaline medium. Intensity of complex is proportional to the amount of calcium. This complex was measured at the absorbance of 570 nm.

Serum osteocalcin

Serum osteocalcin was measured using MicroVue Osteocalcin EIA (Enzyme Immuno Assay) kit in which a mouse osteocalcin binds to sample in serum. This anti-osteocalcin binds to anti-mouse IgG alkaline phosphatase conjugate. On addition of substrate, the amount of OST (Osteocalcin) will be quantified by reading the absorbance at 405 nm.

Statistical analysis

The results were analyzed using SPSS 14.3 which involves paired student's t test with unequal variance. Mean and SD. were calculated for each group and were compared between 2 groups. P < 0.05 was taken as point of minimal significance.

RESULTS

As shown in Figures 1 and 2, Bone differentiation marker, serum osteocalcin and serum bone-alkaline phosphatase were found to be significantly high (P < 0.05) when compared to control subjects. Similarly the level of serum calcium [Figure 3] was statistically significant (P < 0.05). But the serum phosphorus level [Figure 3] was slightly high which was not statistically significant.

Figure 1

Effect of spray paint on level of serum osteocalcin in control and experimental subjects. Each bar represents mean ± SD in each group. A P value <0.05 was considered significant. “a” denotes control versus experimental subjects

Figure 2

Effect of Spray paint on level of serum bone specific Alkaline phosphatase in control and experimental subjects. Each bar represents mean ± SD in each group. A P value <0.05 was considered significant. “a” denotes control versus experimental subjects

Figure 3

Effect of Spray paint on level of serum phosphorus and serum calcium in control and experimental subjects. Each bar represents mean ± SD in each group. A P value <0.05 was considered significant. “a” denotes control versus experimental subjects

DISCUSSION

Bone remodeling and turnover is a process mediated by osteoblast in formation and osteoclast in resorption.[14] The coupling of these two cells in maintaining the bone mass is largely regulated by physical,[15] hormone,[16-18] nutrition[1920] and genetic factors.[2122] Chemicals in spray paint such as Lead,[23] mercury, cadmium,[24] isocyanates,[25] xylene,[26] benzene,[27] ethylbenzene, methylbenzene, Alkylphenol ethoxylate, Formaldehye, phthalates, styrene Butyl benzyl phthalate were studied for its individual effects especially on bone metabolism. These chemicals inflict the cellular processes being xenobiotics[28] or endocrine disruptor. Being xenobiotics these chemicals activate xenosensing receptors (Pregnane X Receptor, Constitutive Androstane Receptor, and Arlhydrocarbon Receptor) for activation of monoygenase pathway which results in reactive intermediates and redox cycling of xenobiotics which results in generation of pro-oxidants. This results in oxidative stress and subsequent modification of critical biomolecules. Phthalates were reported to be an endocrine disruptor.[2930] Being an endocrine disruptor, these spray paint chemicals mimics estrogen and blocks the downstream signaling of estrogen.[31]

In present study, we found the increased serum level of bone specific alkaline phosphatase[32] a marker of bone formation found in the osteoblast. It was reported to be increased in bone disorders.[33] In present study, the level was high in spray painters. Spray paint chemicals induce oxidative stress by release of pro-oxidants due to xenobiotic intermediates which destructs the osteoblast. As alkaline phospatase clings to the membrane of osteoblast, due to increased free radicals more bone specific ALP are released into serum. Serum osteocalcin, being secreted in by osteoblast for bone extracellular matrix which is a biochemical marker for bone turnover and bone formation.[3435] We found an increasing trend in spray painter's serum. As the spray paint constitutents being an endocrine disruptor, results in anti-estrognic effect which promotes ostoclastogenesis. Thus in present study, serum osteocalcin is increased in spray painters when compared to normal subjects. Serum calcium and serum phosphorus being a marker for bone turnover and remodeling[36] in our study we found that the increased calcium and phosphorus may be due to the osteoclastogenesis. Based on these inferences, we speculate that spray paint consitutents stimulate free radical generation in osteoblast and being a anti-estrogen, these chemicals may promote osteoclastogenesis. Thus Rapid bone loss occurs because of disturbance in the balance between bone formation and bone resorption. Based on the results obtained from current study, it was concluded that the spray painters are prone for osteoporosis and fracture risk, as their bone turn over and remodeling occurs at high rate.