Effects of Lead on Haem Biosynthesis and Haematological Parameters in Battery Manufacturing Workers of Western Maharashtra, India

Lead inhibits enzymes of haem biosynthesis and alters haematological parameters of battery manufacturing workers (BMW). The main aim of this study is to know the present status of blood lead (PbB) levels and its effect on haem biosynthesis related parameters such as erythrocytes aminolevulinic acid dehydratase (-ALAD), urinary -aminolevulinic acid (U-ALA) and porphobilinogen (PBG) and haematological parameters of BMW. Material and Methods: Forty BMW from Western Maharashtra, India, having age group between 19-42 years were selected as study group and compared with age matched 38 healthy male subjects (control group). From both group subjects, 10 ml blood sample was drawn by puncturing the anteriorcubital vein and the PbB, erythrocytes -ALAD, urinary -ALA and PBG and haematological parameters were measured by using standard methods. Statistical analysis: Between controls and BMW group was carried out by students ‘t’ test. Blood lead levels of BMW showed significant elevation (p<0.001, 1050%) as compared to controls. Activated ALAD (p<0.001, -58.88 %), non-activated -ALAD (p<0.001, -62.06 %) showed significant decrease and ratio of activated to non-activated -ALAD (p<0.05, 29.26 %) revealed significant increase in BMW as compared to controls. Urinary ALA (p<0.001, 161%) and Urinary-PBG (p<0.05, 45.3%) concentrations showed significant increase in the study group as compared to the control group. In battery manufacturing workers, Hb (p<0.001, -16.67%) PCV (p<0.001,-20.31%) MCV (p<0.05, -4.27%) MCH (p<0.05, -5.66), MCHC (p<0.001, -7.16%) and RBC count (p<0.001, -10.39 %) revealed significant decrease, while a significant elevation was seen in the total WBC count (p<0.001, 20.47%) as compared to the controls. Blood lead levels continue to remain high in BMW, in spite of modern techniques used to reduce the lead exposure which inhibits haem biosynthesis and alters haematological parameters. Keyword: Battery manufacturing workers (BMW); Blood lead; -Aminolevulinic acid dehydratase; Urinary -ALA and PBG, Haematological Parameters Kshirsagar et al 478 J Pharm Chem Biol Sci , December 2015-February 2016; 3(4):477-487 INTRODUCTION Lead is a ubiquitous and versatile metal that has been used by mankind for over 9000 years and is today one of the most widely distributed toxins in the environment. Lead enters in the environment from either natural or anthropogenic sources. Lead is a soft, silvery grey metal, melting at 327.5°C, highly resistant to corrosion, pliable, having high density, low elasticity, high thermal expansion, low melting point, easy workability, easily recycled, excellent antifriction metal, and inexpensive. Due to these properties, lead is used for various purposes. Lead is mainly used in acid batteries, colour pigments, jewellery industries, petrol additives (tetra ethyl and tetra methyl), and ship construction, seams of cans used to store food, soldering water distribution pipes, ceramic glazes, paper industries and printing press. Lead and its compounds can enter the environment at any point during mining, smelting, processing, use, recycling, or disposal [1, 2]. The routes of exposure for inorganic lead are inhalation and ingestion. Lead fumes and soluble respirable dust are almost completely absorbed by inhalation. Adults absorb approximately 15% of an ingested dose through the gastrointestinal (GI) tract in contrast to 50% GI absorption in children. Gastrointestinal absorption is generally inversely proportional to particle size and directly proportional to the solubility of the lead compounds. Dietary factors, nutritional status, and the chemical form of the metal and patterns of food intake affect absorption. Once absorbed, lead is found in all tissues, but eventually > 90% of the body burden accumulates (or is redistributed) into bone, where it remains with a half life of 27 to 30 years. Lead is excreted primarily through the urine (> 90%), lesser amounts are eliminated via the faeces, sweat, hair, and nails. [1-4]. Lead has been shown to cause adverse effects in several organs and organ systems, including the hematopoietic, nervous, renal, cardiovascular, reproductive, and immune and it is also mutagenic. The biological effects of lead depend upon the level and duration of exposure. Lead inhibits enzymes of heme biosynthesis [3, 4]. It affects erythrocyte formation by impairing globulin and heme synthesis and depresses serum levels of erythropoietin. Lead also decreases erythrocyte survival through its inhibition of membranebound Na+-K+-ATPase, resulting in decreased hemoglobin synthesis and anemia in children andadults.[1-3] Early symptoms are often subtle, nonspecific, and/or subclinical, involving the nervous system (restlessness, fatigue, irritability, sleep disturbance, headache, difficulty in concentrating, decreased libido), GI system (abdominal cramps, anorexia, nausea, constipation, diarrhea), or musculoskeletal system (arthralgia, myalgia). Other less common conditions include tremors, toxic hepatitis, or acute gouty arthritis (saturnine gout). In general, the number and severity of symptoms worsen with increasing blood lead levels. A high blood lead level of intoxication may result in delirium, coma, and seizures associated with lead encephalopathy, a life threatening condition [1-4]. Occupational exposure to lead is entirely unregulated in many developing countries, and little monitoring is conducted in developed countries. In battery manufacturing industries the metallic lead is mainly used for the making grids, bearings, and solders. Manufacturing processes are usually manual and involves the release of lead particles and lead oxide that can cause severe poisoning and environmental pollution. Battery recycling is an important source of exposure to inorganic lead vapours, particles, and debris [1-4]. The high blood Lead affects almost all organs and systems and impairs the normal functions of the body, a fact which is well documented in literature, However, we must know the present scenario of blood lead level and its effects on Kshirsagar et al 479 J Pharm Chem Biol Sci , December 2015-February 2016; 3(4):477-487 lead exposed population mainly BMW. Since its noted that now days battery industry owners are using modern techniques to reduce the lead exposure. Hence, the aim of this study is to estimate the blood lead level and to see its effects on haem biosynthesis and haematological parameters of occupational lead-exposed population mainly battery manufacturing workers of Western Maharashtra (India). MATERIAL AND METHODS The study group included non-lead exposed healthy male subjects and lead exposed battery-manufacturing workers of Kolhapur city in the Western Maharashtra state of India. The lead exposed groups consisted of 40 male battery-manufacturing workers (BMW) and the non-lead exposed control group consisted of 38 healthy male subjects. The control group subjects were mainly staff of Krishna Institute of Medical Sciences University, Karad. All the study group subjects were in the range of 19– 42 years of age. All the study and control group subjects were non alcoholic and non smokers. Only healthy male subjects were included and those on medication for minor and major illnesses were excluded. Before blood collection, both study and control group subjects were informed about the study objectives and health hazards of lead exposure and its toxicity and written consent was obtained from subjects of both groups. Demographic, occupational and clinical data were collected by using questionnaire and interviews. Majority of battery manufacturing workers had major complaints of loss of appetite, intermittent abdominal pain, nausea, diarrhea, constipation and myalgia. The socioeconomic status of all subjects of both groups was average. Dietary intake and food habits of all subjects were normal. The experimental protocol was approved by the institutional protocol committee and also ethical clearance was obtained from institutional ethics committee. Utmost care was taken during the experimental procedure according to the Helsinki declaration of 1964 [5]. A blood sample of 10 ml was drawn by puncturing the anticubital vein and 5 ml blood was transferred in tube containing heparin and the rest 5 ml was taken in EDTA bulb for biochemical parameters assays included in the study. All the biochemical parameters were measured by standard methods. Blood lead level was estimated using lead Care II blood lead analyzer.The lead care II system uses an electrochemical technique called Anodic Stripping Voltammetry (ASV) to determine the amount of lead in a blood sample. Blood was mixed with lead care treatment reagent and the red blood cells (RBC) were lysed which release lead that was bound to the RBC wall. A negative potential was applied to the sensor to accumulate lead atoms on the test electrode. The potential is rapidly reversed releasing the lead ions. The current produced was directly proportional to the amount of lead in the sample [6]. Erythrocyte–-Aminolevulinic acid dehydratase (ALAD) was estimated by the method of Julian Chisolan et al [7]. Erythrocyte -ALAD acts on -aminolevulinic acid (ALA) to form porphobilinogen, which is further reacted with modified Ehrlich’s reagent to form pink colored compound measured on spectrophotometer at 555 nm. Hg-TCA solution stops the reaction by precipitating the proteins. ALAD activity was estimated by using the following formula: -ALAD activity (μmol ALA utilized/min/L of erythrocytes)= Net absorbance×100×2×35/ (% Hematocrit×60×0.062) Where, 2 = Conversion factor for ALA to PBG 35 = Dilution factor 60 = Incubation time (min) 0.062 = Micromolar absorptivity of modified Ehrlich’s reagent and PBG chromogen. Kshirsagar et al 480 J Pharm Chem Biol Sci , December 2015-February 2016; 3(4):477-487 Activated and non-activated ALAD ratio (Act/Non-act) was determined.  aminolevulinic acid (ALA) was estimated in urine samples by the method of Osamu W. et al. -ALA reacts with acetylacetone and form pyrrole substance, which reacts with pdimethyl amino benzaldehyde. The colored complex was measured spectrophotometrically at 555nm. The results were measured in mg/L [8]. Estimation of porphobilinogen in urine was estimated by Mauzerall & Granick 1956. Porphobilinogen (PBG) from urine reacts with p-dimethyl aminobenzaldehyde (DMAB, Ehrlich’s reagent) in acid solution to form a red compound, which was measured at 555 nm exactly after 5 minutes and the value were calculated according to Rimington formula [9,10]. Urinary PBG (mg/L) = Optical Density × Numbers of times the urine diluted / 70.85 All the hematological parameters were measured by using fully automated Hematology analyzer Sysmax K-4500. Statistical comparison between controls and battery manufacturing workers groups was done by using Graph Pad Instant Demo-[DATASET-1, ISD] software. Statistical analysis between controls and BMW group was carried out by students ‘t’ test. The mean difference was considered significant at