Effects of Calcium/zinc Alone or Co-supplementation on the Hematological and Biochemical Parameters of Lead-exposed Albino Wistar Rats

Effects of Calcium/zinc Alone or Co-supplementation on the Hematological and Biochemical Parameters of Lead-exposed Albino Wistar Rats

Heavy metals that are toxic abound in the environment. Lead is one of the heavy metals that are widespread in our ecosystem. It is found alongside other heavy metals with relatively low anthropogenic concentrations in all parts of the environment. However, human activities have added exceptionally higher concentrations of these heavy metals into the environment, particularly in areas where the metals are mined, processed and used industrially (Ekanam et al., 2015).

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There are no useful functions that lead plays in the human body; rather its presence in the system tends to bring about toxic effects, no matter the exposure pathway. Every organ system of the body can always be affected by toxicity of lead. Outlined mechanisms for toxicity involve elementary biochemical processes at the molecular level. These processes are the ability of lead to mimic the actions of calcium or put up an inhibition. This may affect calcium dependent or its related processes and to interact with proteins (those with amine, sulfhydryl, carboxyl groups and phosphate are included) (ATSDR, 2005).
Lead as a metal with a high poisoning rate has a component limit of 1.0µg/g. This represents an individual`s maximum daily intake. It’s used as a test benchmark for the production of drugs. Prolonged intake of lead at this concentration can be detrimental to human health (Rieuwerts, 2015). Toxicity of lead is so dangerous that it has the potential of creating irreversible health effect. Lead toxicity primarily affects a number of body functions and organ systems such as the central nervous system, kidney function, liver function and hematopoietic system. This produces serious organ diseases (Kalia and Flora, 2005). Flora et al. (2012) stated that acute toxicity of lead is associated with occupational lead exposure even when it’s not too common, while chronic toxicity (prolonged intake) of lead is quite more common. Detection of blood lead (PbB) levels at 40-60µg/dl describes chronic toxicity. It becomes so serious if no treatment is given as at when due. Frequent vomiting, convulsions, lethargy and encephalopathy are some of the characteristics of chronic lead toxicity (Flora et al., 2006; Pearce, 2007).

Calcium is a metal. The body stores not less than 99% of calcium in the bones and teeth, while the fluids contain the remaining 1%. The mechanism of muscle relaxation and contraction is mostly controlled by calcium. Other biochemical roles of calcium include nerve impulse transmission, transfer of information from one brain cell to another. It also inhibits lead uptake in the gastrointestinal tract and long-term storage of lead in the bones (Mahaffey, 1980).
Zinc shares a similar nature with iron, but their relationship cannot be taken too serious because of its weakness. Zinc competes with lead for absorption in the gastrointestinal tract, but is not too strong like iron and calcium to effectively displace lead, although it appears stronger if taken with lysine. Robert (2010) reported that zinc supplementation has weak effect on blood lead (PbB) levels, although some studies have found strong effects of zinc in combination with other nutrients or chelators on lead. The study of Tandon (2000) on rats reported that becozinc was used to treat lead poisoning with chelating agents. His Findings showed becozinc producing more lead exertion compared to vitamin C (ascorbic acid) or vitamin B1 (thiamin) when taken alone. As a result it was taken as safer alternative to treat lead poisoning.
Another animal studies have suggested that damage to some brain functions can be reduced by the presence of zinc; this may be so because the brain has high zinc concentrations (Robert, 2010). Studies on animals have also shown that lead uptake in the liver, kidney and gut can be reduced by zinc but may cause high impact of lead on the thyroid gland and its roles being noticed on bone resorption (Robert, 2010). The reduction of calcium and magnesium concentrations in organs caused by lead and alcohol can also be ameliorated by zinc (Rantham et al., 2006; Fengyuan et al., 2007). Calcium supplementation may be beneficial in treating toxicity of lead in developing rat brain (Gothipola and Davuljigari, 2014).

STATEMENT OF THE PROBLEM
With increased concern about environmental pollution, the relationship between nutritional deficiencies and toxic metals has been of utmost interest (Jorge et al., 2016). Exposures to lead remain very high as lead is said to be ubiquitous, hence lead toxicity is to be reduced. Calcium and zinc supplementations have been found to reduce blood lead (PbB) level, though some studies have suggested otherwise. In this study, efforts are made to determine how calcium and zinc supplementations would reduce serum lead (PbS) level and improve biochemical parameters in mammals as serum/plasma lead have been found to be the toxicologically active lead fraction (Lamadrid-Figueroa et al., 2007).

AIM OF THE STUDY
To investigate if supplementation with calcium/zinc alone or in combination will reduce serum lead (PbS) level and improve some hematological and biochemical parameters in lead exposed rats.

SPECIFIC OBJECTIVES
1. To determine serum lead, calcium and zinc levels in lead exposed-calcium/zinc supplemented rats.
2. To determine the hemoglobin concentration and packed cell volume in lead exposed-calcium/zinc supplemented rats.
3. To determine liver and renal function parameters in lead exposed-calcium/zinc supplemented rats.
4. To correlate serum lead levels with serum level of calcium and zinc in lead exposed-calcium/zinc supplemented rats.

 

CHAPTER TWO
LITERATURE REVIEW
CHEMISTRY OF LEAD
Lead is an element found in group 4 of the periodic table. Its symbol is Pb and it has an atomic number of 82. It is a post-transition metal that is soft and malleable. When lead is cut afresh, it has a colour that is bluish-white, which upon exposure to air deteriorates to a dull grayish colour. This soft metal is decorated with chrome-silver luster (Polyansky, 1986). The physical and chemical properties of lead include softness, malleability, ductility, high density, poor electrical conductivity, high corrosion resistance and reactions with organic compounds. Its malleability exceeds its ductility (Anderson, 2010).
Lead has four stable isotopes as lead-204, lead-206, lead-207 and lead-208. These many isotopes arise as a result of the atomic number of lead (82) that is even. With this high atomic number, lead ranks the second-heaviest element that occurs in nature in different isotopes that have practical applications because of their stability. The four isotopes of lead can undergo radioactivity (alpha decay) to form isotopes of mercury which may be accompanied by the release of energy, although no observation has been made for any of them (Audi et al., 2003).
A total 38 lead isotopes have been produced. They are those between the atomic masses of 178 and 215 (Audi et al., 2003).

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The most stable radioisotope of lead is lead-205 which has a half-life of about 1.5×107 years. It is seconded by lead-202 (a synthetic isotope) with a half-life of 5.3×104 years. Lead-202 is longer than all the natural trace radioisotopes. Characterization has been done on about 47 nuclear isomers that corresponded to 24 isotopes of lead isotopes. Lead-204m2 is the longest lived isomer. Its half-life is around 66minutes (Audi et al., 2003).
Two main oxidation states of lead are +2 and +4. The oxidation state of +4 is common for the group 4, while the stability of +2 increases down the group. It does not exist for carbon and silicon, it is greatly minor for germanium, it is good for tin, and is much better for lead. Oxidizing agents like oxygen and fluorine can only oxidize lead to lead (II) ion, Pb2+ initially even when they are termed the strongest elements of oxidation (Greenwood and Earnshaw, 1998). This is caused by their relative effects, specifically, the effect of the inert pair which shows up when a large electronegativity difference occurs between lead and the anions such as oxide, halides, nitrides. At this juncture, lead is being positively charged which leads to a stronger contraction of the 6S orbital than the 6p orbital, thereby producing inert 6S orbital in ionic compounds (Sanshiro, 1997).
Lead burns with a bluish-white flame when grinded (powdered). Finely grinded lead exhibits pyrophoricity as it is with many other metals (Charles et al., 1966). When lead is released to the atmosphere in bulk, an insoluble lead oxide is formed as layer of protection which prevents the metal from reacting further. If lead is exposed to another area, maybe a chemical environment, sulfate, chloride or any other insoluble compound can also form the protective layer (Rieuwerts, 2015).
Lead is employed in the production of materials such as shot, bullets, lead-acid batteries paints, weights, etc. It is also used as part of solders, fusible alloys, as a radiation shield and in construction of buildings. All these products are poisonous to humans and animals whether inhaled or ingested. Lead toxicity has become a major risk factor to public health, especially in countries that are still developing. For this, various measures on public and occupational health care are being taken to control lead exposure, even when there are still reported cases of lead intoxications (Flora et al., 2012).

SOURCES OF LEAD EXPOSURE
Lead is everywhere in the environment. It is one of the earliest metals discovered by man. The unique physical and chemical properties of lead have given rise to its widespread usage in the production of paints, plastics, automobiles, ceramics, etc. This involvement of lead in different industries has resulted to a huge release of free lead in biological systems and the ecosystem (Flora et al., 2012).
There are no useful functions of lead in the biological system; its presence brings about toxic effects, no matter the route of exposure. It is a potent occupational toxin and its toxicological outcomes cannot be over emphasized. The sole reason for leads prolonged persistence in the environment is its inability to be biodegraded. Human exposure to lead by humans happens via many sources like paints made of lead, lead smelting, leaded gasoline, batteries, bearings, lead pipes, coal combustion, etc. (Flora et al., 2012)
The main source of lead exposure is ingestion of lead-based paints.

Lead-based paints peel and pulverize into dust during deterioration and then move into the body via hand-to-mouth contact or they contaminate water, food or alcohol (Rieuwerts, 2015). Exposure to lead or its compounds can also occur when some home remedy drugs are taken (Rieuwerts, 2015). Lead can enter the body through eating of fruits and vegetables that may have been contaminated by its high levels in the soils they were cultivated, for instance, plants cultivated in dumpsites (Obasi et al., 2015). Water pipes made of lead pose a problem to people who live in areas where water is acidic or soft because lead is dissolved in the acidic water. Insoluble layers are formed by hard water in the pipes (Moore, 1977).
Another source of lead exposure is inhalation. Occupational workers are mostly affected. At most the body absorbs about 20-70% ingested lead and all inhaled lead. Children are more susceptible to lead exposure and they tend to absorb and retain more lead than adults (Rieuwerts, 2015).
Exposure of lead through the skin (dermal exposure) is mostly experienced by few people who work with organic lead compounds. It does not really put up a high concern to the general public, as the use of leaded gasoline has been stopped since 2007 by many countries. It takes the skin more time to absorb inorganic lead (Rieuwerts, 2015).
The soils and waters have been seriously contaminated as lead is being extracted, produced, used and disposed, thereby intoxicating the living organisms. Srivastav et al. (2013) studied and reported the effects of lead on the plasma electrolytes of freshwater fish. In as much as the use of leaded petrol and gasoline has been stopped by most countries, persistence of high concentrations of lead still occurs in soils especially in urban and post-industrial settings. On the hand, many countries of the world still experience industrial lead emissions (Rieuwerts, 2015).

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EFFECTS OF LEAD IN BIOLOGICAL SYSTEMS
Lead toxicity is so hazardous that it can cause health effects that are irreversible. It interferes with a number of body functions and primarily affects the renal, hepatic, nervous and hematopoietic systems to cause serious diseases (Kalia and Flora, 2005).
Lead interferes with a variety of enzymes. This is the primary cause of its toxicity as many enzymes allow it to bind on their sulfhydryl groups (Pearson and Schonfeld, 2003). Part of toxicity of lead comes as a result of its ability to mimic other metals (cofactors) that are involved in the biological processes so as to displace them at the interaction site of the enzymes on which they act (Dart et al., 2004). Lead does not function properly as cofactor because of its differing chemistry but has the ability to disrupt enzymes normal reactions. Some of the essential metals that lead mimics are calcium, zinc and iron. Kosnett, (2006) and Venugopal, (2013) observed that high levels of calcium and iron give protections against lead poisoning and at low concentrations living organism are rendered more susceptible to lead poisoning. The body absorbs very fast and efficiently lead salts, its accumulation results to both acute and chronic lead poisoning (Venugopal, 2013).
Lead poisoning is a risk factor for public health, especially in developing countries. Exposure to lead produces various deleterious effects on the following organ systems.

Effect of Lead on Nervous System
Lead exposure has great effects on both the central nervous system and the peripheral nervous system. This happens because the nervous system provides a very sensitive target site for lead. In adults, the effect of lead is mostly on the peripheral nervous system while in children, the effect is more on the central nervous system (Brent, 2006; Bellinger, 2004). Children show more neurological lead effects, although no harm is caused when the level of exposure is within the limit (<10kg/dl) (Canfield, 2003). Flora et al. (2012) reported that the developing nervous system absorbs more lead which leads to the unborn and new born babies being more susceptible to neurological lead effects. The direct impact of lead exposure is encephalopathy. Some of its clinical symptoms are fatigue, memory loss, head-ache, dullness, poor attention span, decreased libido, muscular tremor, loss of appetite, etc. At very high exposures, lead produces more severe symptoms such as lack of coordination, paralysis, delirium, convulsions, coma, etc. (Flora et al., 2006).

Lead gains more access to the brain of children than adults (Needleman, 2004). At low levels of lead exposure, children may be hyperactive, irritable or inattentive. Permanent brain damage and death can occur at high levels of lead exposure (Cleveland et al., 2008).
Lead exposure on the peripheral nervous system produces peripheral neuropathy which is characterized by reduced motor activity as a result of myelin sheath loss. The transduction of nerve impulses is impaired when the nerve is not insulated by myelin sheath. This brings about fatigue, muscular weakness and lack of muscular co-ordination (Sanders et al., 2009).

Effect of Lead on Renal Function
Association between lead exposure and kidney effects has been undertaken by many studies (Missoun et al., 2010). It has been reported that lead toxicity causes kidney functions impairment as one of its most silent characteristics (Chang et al., 1980). Glycosuria, hyperphosphaturia and aminociduria are the results of proximal tubular function impairment by acute lead toxicity on high dose. However, as exposures continue, toxic stress may occur on the kidney, if not treated, may degenerate to irreversible chronic lead nephropathy. Grant (2008) reported that high levels of lead exposure (>60µg/dl) produced renal dysfunction but lower levels produced mild damage.
Proximal tubular nephropathy, glomerular sclerosis and intestinal fibrosis are the characteristic features of lead nephropathy (Diamond, 2005; Loghman-Adham, 1997). Tubular transport mechanism impairment is a functional feature of acute nephropathy. It is also associated with morphological changes found on the tubular epithelium together with the appearance of nuclear inclusion bodies which contains lead protein complexes. It does not lead to the appearance of protein in the urine but can lead to the excretion of amino acids, glucose and phosphates, a clinical condition known as Fanconi’s syndrome (Rastogi, 2008).

Effect of Lead on Reproductive System
Many effects on the reproductive system in humans have been caused by lead exposure. Some of the symptoms of effects of lead in men are infertility, abnormal function of the prostate, reduced libido, abnormal spermatogenesis, changes in serum testosterone, etc. Infertility, miscarriage, premature delivery, premature membrane ruptures and pregnancy hypertension are some of the effects of lead on the reproductive system of women (Flora et al., 2011). It has been reported that lead has direct effect on the fetus during the stages of development (Saleh et al., 2009).
It has also been found that high blood lead ( PbB) levels affect pregnancy and its outcomes (Ugwuja et al., 2013), but at low levels, its effect is not well understood. Some studies have suggested that pregnancy complications may arise when the blood lead levels go beyond 10µg/dl. This can lead to reduction of gestation length, miscarriage, increased risk of gestational hypertension and preterm delivery (Vigeh et al., 2006). Vigeh et al. (2004) and Sowers et al. (2002) reported that even at blood lead level of 5µg/dl, adverse pregnancy outcomes were still recorded. The study of Anttila and Sallmen (1995) illustrated that, more miscarriages are recorded by women whose male partners are exposed to lead. Higher rates of congenital epilepsy and cardiovascular diseases have been observed in children whose parents are occupational lead workers (Hu et al., 1992). Women with blood lead levels of 5-9µg/dl were 50-60% more vulnerable to spontaneous abortion than were those whose blood lead levels were less than 5µg/dl (Borja-Aburto et al., 1999).

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Effect of Lead on Hematopoietic System
The ability of the body to synthesize hemoglobin is seriously affected by lead. Lead has a way of upsetting the enzymatic steps involved in the pathway of heme metabolism. It makes cell membrane more fragile thereby reducing the life span of erythrocytes in circulation to cause anemia (Guidotti et al., 2008; Cornelis, 2005). Two types of anemia caused are hemolytic and frank anemia. The former develops when there is acute high-levels of lead exposure, while the later occurs only when the blood lead level is so high for a prolonged period of time (Vij, 2009).
Specifically, lead decreases the synthesis of heme by interfering with activities of d-aminolevulinic acid dehydratase (ALAD) and ferochelatas. It also inhibits activity of aminolevulinic acid synthetase (ALAS), an enzyme of the mitochondria that increases the rate of aminolevulinic acid (ALA) formation. Ferrochelatase catalyzes the incorporation of iron into protoporthyrin to produce heme (Piomeli, 2002).

The three above mentioned enzymes are all inhibited by lead but its effect is more pronounced on ALAD which has been a clinical tool to determine the degree of lead poisoning. The accumulation of aminolevulinic acid detectable in both plasma and urine is a result of the inhibition of ALAD. The presence of aminolevulinic acid in urine or plasma can occur even when the blood lead levels are less than 10µg/dl. Although inhibition is first detected when blood lead levels are between 10-20µg/dl. The synthesis of heme begins to decrease if 80-90% activity of ALAD has been inhibited. This occurs at lead concentration that is as high as 55µg/dl (Ahamed et al., 2005).
The mechanism of action that leads to the shortening of the erythrocytes life cycle is not clear. Earlier effects of lead observed on hematology had shown the presence of dense material in red blood cells. This is also clinical tool to detect lead poisoning. These materials are products of ribonucleic acid (RNA) catabolism (Patrick, 2006).

Effect of Lead on Cardiovascular System (Hypertension)
Hypertension is a clinical condition that is caused by different agents. Some of the risk factors for hypertension are diet, weight, age and physical exercise habits. Exposure to lead can cause hypertension at the onset. Both acute and chronic lead poisoning produces cardiac and vascular disorder which may cause death (Navas-Alien et al., 2007). Although low to moderate lead level exposures (PbB level < 30µg/dl) contribute little the development of hypertension, while higher exposures create room for hypertension. Hypertension caused by low level lead can be seen in living organisms (ATSDR, 2005).
There are other disorders of lead effect on cardiovascular system. They are ischemic coronary heart disease, peripheral vascular disease and cerebrovascular accidents. There is evidence that lead exposure has a relationship with hypertension but it is mostly applied when toxicity of lead is on cardiovascular outcomes (Navas Alien et al., 2007).

Effect of Lead on Bones
Lead has effects on the development and health of bones. The body uses bones as its primary site to store lead. (Renner, 2010; Silbergeld et al., 1993). Lead is stored in both the exchangeable non-exchangeable pools of the bone. It moves into the plasma at so easily from the exchangeable pool but leaves the non-exchangeable pool and goes to the surface during bone active reabsorption (Patrick, 2006). Many factors such as lead exposure rate, age, pregnancy, gestation and race determine the mobilization and storage of lead in the bones (Flora et al., 2012).

 

MECHANISM OF LEAD TOXICITY
Lead as a heavy toxic metal has been widely studied. The toxicological effects caused by lead in the system come from the mechanisms of various cellular, intracellular and molecular activities (Flora et al., 2012).

Oxidative Stress: This is caused when the production of free radicals and the ability of the system to readily detoxify the reactive intermediates are not balanced or when a resulting damage is not repaired (Flora, 2011). Oxidative stress is a major mechanism of lead induced toxicity according to reports. During lead induced toxicity, oxidative stress produces two different pathways that work simultaneously. The first pathway initiates the generation of Reactive Oxygen Species (ROS) eg. Hydroperoxides (HO2), while the second pathway leads to the depletion of the antioxidant reserves (Flora, 2002).

Generated ROS is removed by antioxidant. Glutathione (GSH) is the most significant antioxidant found in the animal cells. It is used to remove free radicals (Mates, 2000). Glutathione can exist in reduced (GSH) or oxidized state (GSSG). Glutathione in a reduced state donates reducing equivalents (H+ + e-) from its thiol groups present in cysteine residues to ROS to ensure stability. In an oxidized state, it combines with another molecule of glutathione to form glutathione disulfide (GSSG) with the help of an enzyme called glutathione reductase (GR).

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