Elevated blood Lead impairs the Hematological and Heme Biosynthesis related parameters of Silver Jewellery Workers

To see the present scenario of blood lead (BPb) levels of Silver JewelleryWorkers (SJW) and its effects on haematological and heme biosynthesis parameters. Forty-two SJW of having an age range of 20-45 years were included for this study and comparedwith 50 healthymale subjects of the same age. Blood lead, RBC δaminolevulinic acid dehydratase (δ-ALDH), urinary δaminolevulinic acid (Uδ-ALA), urinary porphobilinogen (U-PBG) and haematological parameters were measured. Blood lead level of silver jewellery workers was signi icantly elevated (p<0.001, 325%) and non-activated δ-ALAD (p<0.01), activated δ-ALAD (p<0.05) were notably reduced and the ratio of A/NA δ-ALAD (p<0.01)was considerably enlarged in SJW as compared to control group subjects. Urinary excretion of δ-ALA (p<0.001) and PBG (p<0.001) levelswere extensively increased in SJWas compared to subjects from the control group. Hb (p<0.001), PCV (p<0.001), MCV (p<0.001), MCH (p<0.05) and RBC (p<0.001) were notably reduced in SJW, while total WBC count (p<0.001, 29.8%) was considerably elevated as compared to subjects from the control group. Conclusions: Increased BPb level in SJW indicates themore absorption of lead from the GIT,which inhibits heme biosynthesis, and alters haematological parameters.


INTRODUCTION
Lead is a multipurpose metal present everywhere and occurring naturally in the earth's crust and has been used by humans for over 9000 years. It is an inexpensive, soft, silvery grey metal with a low melting point of 327.5 • C. Its properties include high resistance to corrosion, pliability, high density and thermal expansion, low elasticity, easy workability, and recyclability, with excellent antifriction capabilities. Due to these properties, lead inds usage in a variety of manufacturing processes like acid batteries, cable sheathing, ceramic glazing, soldering, and ship construction. Lead, and its alloys ind usage in pipes, cable covers, shot and ammunition, and radiation shields. Lead compounds are used as pigments in paints and dyes, due to its anti-corrosive properties, Tetra-methyl and tetraethyl lead was used in gasoline additives as antiknock agents and to enhance octane rating. Lead, along with other metals like mercury and arsenic, also inds its usage in cosmetics and traditional complementary and alternative medicine in India (Ayurveda) and China (ATSDR, 2017).
Lead after absorption through the respiratory and digestive systems, gets deposited in all tissues with maximal (>90%) accumulation found in bones. Halflife of lead in blood and soft tissue is 30-45 days and in bones 27-30 years. It is primarily excreted through urine (90%) with small amounts found in faeces, sweat, hair, and nails. Depending on the blood lead level and duration of exposure, lead can cause widespread adverse health effects on numerous organ systems including hematopoietic, nervous, renal, reproductive, cardiovascular and immune systems. Three enzymes known as δ-aminolevulinic acid dehydratase, coproporphyrin oxidase, and ferrochelatase from heme biosynthesis, are inhibited by increased blood lead and also impairs the globulin synthesis, increased blood lead decreases serum levels of erythropoietin hormone and impairs the erythrocyte formation. Lead decreases erythrocyte survival rate by inhibiting the membrane-bound Na+/K+-ATPase, which results in impaired haemoglobin formation. Inhibition of heme biosynthesis by lead results in a reduction of heme pool and affect the nervous, renal and hepatic systems (ATSDR, 1998).
The occupational exposure to lead in developing countries like India is entirely unregulated. Workers employed in silver jewelry industries are required to perform activities like silver distillation from waste through smelting and alloying; manufacturing the silver wires, cutting, assembling, designing, polishing, and plating of silver rings and jewellery (ATSDR, 2007). These activities are usually performed at unregulated shops and homes without adequate training and protective gear, thereby potentially exposing them to toxic levels of lead fumes and dust. These also pose a health hazard for people, especially children, living near these shops.
Keeping these points in perspective, we conducted this study to understand the level of lead exposure among silver jewellery manufacturing workers from the unorganised sector of Maharashtra, India by ascertaining their blood levels of lead and its adverse effects on heme biosynthetic pathway and haematological parameters.

MATERIALS AND METHODS
Forty-two male silver jewellery manufacturing workers from village Hupari (Kolhapur) situated in Western Maharashtra, India were enrolled in this study as a study group. And for comparison, 50 non-lead exposed healthy male subjects from the same area were included as a control group. The subjects of study and control groups were recruited after satisfying the inclusion criteria. The age range of both group subjects was 20-45 years. Those who are on medication for signi icant diseases were excluded from this study. Silver jewellery manufacturing workers with a history of lead exposure more than ive years were enrolled in this study. The study and control group subjects were on the same dietary intake, food habits, and socioeconomic status. From both group subjects, the occupational, clinical and demographic data were collected by taking their interviews during blood collection time and by using pre-structured questionnaires. Before enrolling study and control groups subject, the informed consent was collected, and all the study participants were informed about the health hazards of lead exposure. Ethical study approval [2016-2017/075] was taken, and all the study was carried out as per guidelines of the 1964 Helsinki Declaration (World Health Organization, 1995).
From study and control group subjects, the 10 ml blood was collected in EDTA bulb and a tube containing heparin for quanti ication of blood lead levels, haematological and heme biosynthesis-related investigations. Blood lead analyser special instrument, manufactured by Magellan Diagnostics, USA was mainly used for accurate quanti ication of blood lead levels. The principle of this instrument is based on Anodic Stripping Voltammetry. Lead care treatment reagent was used to lyse the red blood cells and releases the lead particles. These particles then accumulate on the test electrode. Then a negative potential was applied to the sensor. The potential was quickly reversed, which releases the lead particles. The current formed was directly proportional to the number of lead particles in the blood (Patil et al., 2006a). The δ -aminolevulinic acid dehydratase (δ -ALAD) from red blood cells was determined by using the Julian Chisolan et al. method (Patil et al., 2006b). On δ-aminolevulinic acid (δ-ALA) substrate, the enzyme δ -aminolevulinic acid dehydratase (δ -ALAD) acts and form porphobilinogen (PBG), which further reacted with Ehrlich's reagent to give pink coloured complex. The intensity of the colour was measured at 555 nm on a spectrophotometer.
Urinary δ-aminolevulinic acid (U δ-ALA) was determined by using the Osamu et al. method (Declaration of Helsinki, 1964). U δ-ALA reacts with acetylacetone to forms pyrrole, which then reacts with Ehrlich reagent to give red colour. This compound was extracted with chloroform, and the colour intensity was measured spectrophotometrically at 555 nm. Urinary porphobilinogen (U-PBG) was determined by using Mauzerall and Granick method (Magellan Diagnostics, 2015). U-PBG reacts with P-dimethyl amino benzaldehyde in acidic condition to form a red complex, the colour intensity was measured at 555 nm precisely after ive minutes and PBG were calculated by using Rimington formula (1958) (Chisolm et al., 1985).
Haematological parameters were estimated by using fully automated Hematology analyser Sysmex (Wada et al., 1969). Statistical comparison between heme biosynthesis and haematological parameters of controls and silver jewellery workers was made by Student's t-test using Instat GraphPad software.

RESULTS
Blood lead levels of silver jewellery manufacturing workers were signi icantly elevated, and red blood cells non-activated δ-ALAD, activated δ-ALAD were notably reduced. The ratio was considerably enlarged in SJW as compared to control group subjects. Urinary excretion of PBG and δ-ALA were extensively elevated in SJW when compared with controls.
Signi icant negative correlation between blood lead with activated δ-ALAD, non-activated δ-ALAD and positive correlation with Act / N-Act δ-ALAD ratio, U-δ-ALA and U-PBG were observed in the study group.
Hematological parameters such as Hb, PCV, MCV and RBC count were notably reduced in silver jewelry workers, while total WBC Count was considerably elevated as compared to control.
A signi icant negative correlation between blood lead level with Hb, HCT, MCV, RBCs and a positive correlation with WBCs were observed in the study group.

Discussion
Blood levels of lead among subjects belonging to the study group were notably elevated (325%, p<0.001) when compared with the controls shows that the absorption of lead from the gastrointestinal tract was more in silver jewelry manufacturing workers. Such increased levels have also been described in another study (ATSDR, 2007). In the silver jewellery industry, lead is mainly used for making silver rings, an ornament, usually worn around ankles/toes. Lead melts at high temperatures which are then poured in 3 feet mold thereby making lead wires. These wires are then stretched to 15 feet by machine and coated with silver plating. When reheated, lead melts leaving behind silver wires which are made into rings. The molten lead is then reused. The lead smelting process releases fumes/vapors which are inhaled by workers and their family members, considering most of these work are done in their household without adequate protective gear.
Absorbed lead is mainly excreting in feces and urine and very less quantity of lead is excreting through, saliva, sweat, breast milk, nails and hair (Mauzerall and Granick, 1956). It gets accumulated in soft tissues and bones. The ninety-eight percent of lead in erythrocytes is mainly bound to proteins within the cell and roughly 40-75% of lead is bound with albumin (Rimington, 1971). The clinical symptoms including fatigue, peripheral neuropathy, encephalopathy, abdominal colic, dyspepsia, anemia, and have been reported in the literature due to increased blood lead levels (ATSDR, 2017).
Red blood cells non-activated δ-ALAD (-21.28%, p<0.01) and activated δ-ALAD (-18.47%, p<0.05) were considerably reduced and A /NA ratio of δ-ALAD (9.6 %, p<0.01) were notably elevated in the study group when compared with controls (Table 1 and Figure 1). The decreased activity of red blood cell δ-ALAD might be due to lead. Lead inhibits this enzyme by binding to sul hydryl group of δ-ALAD. Earlier studies reported that lead inhibits 3 enzymes of heme biosynthesis pathway i.e. δ-aminolevulinic acid dehydratase, coproporphyrin oxidase, and ferrochelatase (Trester, 1999). Our results are similar to the earlier study, reported in the literature (Stauber et al., 1994).
The urinary δ-ALA (124%, p<0.001) and urinary-PBG (53%, p<0.001) excretion were notably elevated in the silver jewellery manufacturing workers when compared with the controls, which might be because of inhibition of enzymes of heme biosynthesis pathway. These indings also consistent with an earlier study (Al-Modhefer et al., 1979).
The adverse effects of lead on the hematopoietic system in both humans and animals, resulting in increased urinary excretions of porphyrins, coproporphyrins, δ-aminolevulinic acid, erythropoietic protoporphyria, zinc protoporphyrin, and anemia has been well documented in the literature (Trester, 1999). A noticeable interference with heme biosynthesis pathway enzymes leads to reduction in the Hb concentration in blood. Our results are also consisting with another study (EPA, 1986). Impaired erythropoietin hormone production due to renal damage, resulting in insuf icient maturation of erythroid cells also have been suggested as a causative mech-

Figure 1: Percentage Change of blood investigations of SilverJewellery Workers with respect to Control
anism for lead-induced anemia (Osterode et al., 1999).
However, in our study alterations of hematological parameters were not severe which might be due to slightly elevated blood lead level (Mean PbB of SJW is 23.23 µg/dl and PbB range 11.9 -37 µg/dl). Similar results in an earlier study support our indings (ATSDR, 2007). The increased WBC count (p<0.001, 29.8%) in our study may be due to the dust particles and vapors of lead. All the silver jewellery workers were working in unhygienic/unventilated places and also were exposed to the high temperature, thereby leading to increased WBC count.

Figure 2: Percentage Change of Hematological Investigations of Silver Jewellery Workers concerning Control Group
it appears that the impairments of hematological and heme biosynthesis-related parameters because of increased lead were consistent among 29-49% of silver jewellery workers.
Similar blood levels of lead and its adverse impact on hematological and heme biosynthesis related parameters may also be found in workers involved in many unorganized industries involved in battery recycling and production of leaded glass and pottery (ATSDR, 2017).

CONCLUSION
This study found signi icantly higher levels of lead among silver jewellery workers, thereby negatively impacting their hematological parameters. This inding might be due to more occupational exposure to lead fumes and vapors. Therefore, the silver jewellery manufacturing workers should be regularly screened for blood lead levels and its health hazards. Also, these workers should be motivated to use protective gear such as masks, goggles, aprons, etc during work to minimize the adverse effect of lead exposure.