Heavy metals are defined as metallic elements that have a relatively high density compared to water. In recent years, there has been an increasing ecological and global public health concern associated with environmental contamination by heavy metals. Human exposure has risen dramatically as a result of an exponential increase of their use in several industrial, agricultural, domestic and technological applications. Sources of heavy metals in the environment include geogenic, industrial, agricultural, pharmaceutical, domestic effluents, and atmospheric sources. With the assumption that heaviness and toxicity are inter-related, heavy metals, such as lead, are able to induce toxicity at low level of exposure. In human beings, lead has no biological functions and once it enters the body, it causes severe health effects (ATSDR, 2007).
Lead is a heavy, bluish gray, low melting metal that occurs naturally in the Earth’s crust. However, it is rarely found naturally as a metal. It is usually found combined with two or more other elements to form lead compounds (Sparks, 2017). Many of its physical and chemical properties such as softness, malleability, ductility, poor conductibility and resistance to corrosion, have favored that man uses lead and lead compounds since ancient times for a great variety of applications (Hernberg, 2000). Lead is easily molded and shaped. For these reasons, lead has been used by humans for millennia and is widespread today in products as diverse as pipes, storage batteries, pigments and paints, glazes, vinyl products, weights, shot and ammunition, cable covers, radiation shielding etc. Tetra ethyl lead was used extensively from the 1930s to the 1970s as a petrol additive to improve engine performance (WHO, 2010).
Sources of Lead:
Environmental levels of lead have increased more than 1,000 fold over the past three centuries as a result of human activity (ATSDR, 2007). Lead can enter the environment through releases from mining lead and other metals, and from factories that use lead, lead alloys or lead compounds. Lead is released into the air during burning coal, oil or waste. Once lead gets into the atmosphere, it may travel long distances if the lead particles are very small. Lead is removed from the air by rain and by particles falling to land or into surface water. Sources of lead in dust and soil include lead that falls to the ground from the air, and weathering and chipping of lead based paint from buildings, bridges, and other structures. Landfills may contain waste from lead ore mining, ammunition manufacturing, or other industrial activities such as battery production (Carocci et al., 2016; Patrick, 2006; Vorvolakos et al., 2016). Past uses of lead such as its use in gasoline are a major contributor to lead in soil, and higher levels of lead in soil are found near roadways. Most of the lead in inner city soils comes from old houses with paint containing lead. Lead may also enter foods if they are put into improperly glazed pottery or ceramic dishes and from leaded crystal glassware (ATSDR, 2007; Carocci et al., 2016).
Uses of Lead:
The Romans were the ﬁrst to use lead on a large scale in the manufacture of pipes for water supply, manufacture of tableware and kitchen utensils or even as pigment. Lead acetate was used later as a sweetener for wine and cider as well as in medicine for treating several diseases. Lead poisoning was very important due to its widespread use in pottery, pipes, boat building, manufacture of windows, arms industry, pigments and printing of books. Many of these uses declined or disappeared throughout the 19th century, but also introduced new ones during 19th century, as its application for improving the octane rating of gasoline by the addition of tetraethyl lead, its use in glass containers for cooking or the use of paint with lead compounds (Carocci et al., 2016; García-Lestón et al., 2010). Lead compounds are used as a pigment in paints, dyes, and ceramic glazes and in caulk. The amount of lead used in these products has been reduced in recent years to minimize lead’s harmful effect on people and animals. Nowadays, although the vast majority of its uses have disappeared, lead is still present in many industrial activities such as car repair, manufacturing and recycling of batteries, lead paint removal, demolition, reﬁning, ceramic glaze, brass and smeltery (Carocci et al., 2016; NRC, 1993; Patrick, 2006; Vorvolakos et al., 2016). It is also used for maintenance of structures found in the open air as bridges or water towers, in solders of cans of food or beverages, glazed ceramic, and can also be present in drinking water or in tobacco smoke (Carocci et al., 2016; García-Lestón, 2010). Lead paint is a primary source of lead exposure and the major source of lead toxicity. Lead is also found in lead-glazed ceramics; and food eaten or stored in containers painted with lead-based paint or lead-containing glaze may contain significant amounts of lead (Patrick, 2006). Herbal remedies from India, China, and other parts of Asia may be potential sources of lead exposure. Certain Ayurvedic herbal products manufactured in South Asia were found to be contaminated with it. Lead compounds are found in some cosmetics, such as surma and kohl. Some types of hair colorants, cosmetics, and dyes contain lead acetate (ATSDR, 2007).
Lead has been used since ancient times and that it is a non-biodegradable element, environmental pollution caused is persistent and widespread, affecting the population at large. Lead is unique in that man-made sources contribute almost solely to exposure in the post-industrial era. So, lead poisoning has been known to men, but the situation worsened in the 18th century with the industrial revolution. People may be exposed to lead when they work in jobs where lead is used. Lead exposure occurs during the manufacture of ammunition, batteries, sheet lead, solder, some brass and bronze plumbing, ceramic glazes, caulking, radiation shields, circuit boards, military equipment (jet turbine engines, military tracking systems) and some surgical equipment. People who work in lead smelting and refining industries, brass/bronze foundries, rubber products and plastics industries, soldering, steel welding and cutting operations, battery manufacturing plants and lead compound manufacturing industries may be exposed to lead (Carocci et al., 2016; Patrick, 2006). Construction and demolition workers and people who work at waste incinerators, pottery and ceramics industries, radiator repair shops, and other industries that use lead solder may also be exposed. Painters who scrape old paint may be exposed to lead in dust. Families of workers may be exposed to higher levels of lead when workers bring home lead dust on their work clothes (ATSDR, 2007).
Absorption and Distribution of Lead in Body:
Some of the lead that enters in the body comes from breathing in dust or chemicals that contain lead. Once this lead gets into the lungs, it goes quickly to other parts of the body through blood. Larger particles that are too large to get into the lungs can be coughed up and swallowed. One may also swallow lead by eating food and drinking liquids that contain it. The amount that gets into the body from the stomach partially depends on when an individual ate the last meal. It also depends on age and how well the lead particles dissolved in the stomach juices (ATSDR, 2007). Children absorb about 50% of ingested lead. Whereas, experiments using adult volunteers showed that, for adults who had just eaten, the amount of lead that got into the blood from the stomach was only about 6% of the total amount taken in. In adults who had not eaten for a day, about 60-80% of the lead from the stomach got into their blood. In general, if adults and children accept the same amount of lead, a bigger proportion of the amount swallowed will enter the blood in children than in adults (ATSDR, 2007; Carocci et al., 2016).
Dust and soil that contain lead may get on the skin, but only a small portion of the lead will pass through the skin and enter in the blood if it is not washed off. One can, however, accidentally receive lead that is on hands by eating, drinking, smoking, or applying cosmetics. More lead can pass through skin that has been damaged (for example, by scrapes, scratches, and wounds). The only kinds of lead compounds that easily penetrate the skin are the additives in leaded gasoline, which is no longer sold to the general public. Therefore, the general public is not likely to encounter lead that can enter through the skin (ATSDR, 2007).
Shortly after lead gets into the body, it travels in the blood to the soft tissues and organs (such as the liver, kidneys, lungs, brain, spleen, muscles, and heart). After several weeks, most of the lead moves into the bones and teeth. In adults, about 94% of the total amount of lead in the body is stored in the bones and teeth. About 73% of the lead in children’s bodies is stored in their bones. Some of the lead can stay in the bones for decades; however, some lead can leave bones and re-enter in the blood and organs under certain circumstances (e.g., during pregnancy and periods of breast feeding, after a bone is broken and during advancing age). Body does not change lead into any other form. Once it is taken in and distributed to the organs, the lead that is not stored in the bones leaves body in the form of urine or feces (ATSDR, 2007).
Lead in Red Blood Cells:
Lead in blood is rapidly taken by red blood cells, where it binds to several intracellular proteins. Although the mechanisms by which lead crosses cell membranes have not been fully elucidated, results of studies in intact red blood cells and red blood cell ghosts indicate that there are two, and possibly three, pathways for facilitated transfer of lead across the red cell membrane. The major proposed pathway is an anion exchanger that is dependent upon HCO3 and is blocked by anion exchange inhibitors (ATSDR, 2007). A second minor pathway, which does not exhibit HCO3 dependence and is not sensitive to anion exchange inhibitors, may also exist (Simons, 1986). Lead and calcium may also share a permeability pathway, which may be a Ca2+ channel. Lead is extruded from the erythrocyte by an active transport pathway, most likely a (Ca2+, Mg2+)-ATPase (Calderón-Salinas et al., 1999).
Delta-Aminolevulinic acid dehydratase (ALAD) is the primary binding ligand for lead in erythrocytes (Bergdahl et al., 1998; Gonick, 2011). Lead binding to ALAD is saturable; the binding capacity has been estimated to be approximately 850 μg/dL red blood cells (or approximately 40 μg/dL whole blood) and the apparent dissociation constant has been estimated to be approximately 1.5 μg/L (Bergdahl et al., 1998). Two other lead binding proteins have been identified in the red blood cell, a 45 kDa protein and a smaller protein(s) having a molecular weight <10 kDa (Bergdahl et al., 1997, 1998). Strong binding capacity of lead for sulfhydryl proteins creates interference with various enzymes and structural proteins. The most well-known of these distortions involves interference with the heme synthetic pathway, specifically the ALAD enzyme. Of the three principal lead binding proteins identified in red blood cells, ALAD has the strongest affinity for lead and appears to dominate the ligand distribution of lead (35–84% of total erythrocyte lead) at blood lead levels below 40 μg/dL (Bergdahl et al., 1998; Sakai et al., 1982; 2000). Lead binds to and inhibits the activity of ALAD (Gercken and Barnes 1991; Gibbs et al., 1985; Sakai et al., 1982, 2000;). Synthesis of ALAD appears to be induced in response to inhibition of ALAD and, therefore, in response to lead exposure and binding of lead to ALAD (Fujita et al., 1982; Boudene et al., 1984).
Several mechanisms may participate in the induction of ALAD, including (1) inhibition of ALAD directly by lead; (2) inhibition by protoporphyrin, secondary to accumulation of protoporphyrin as a result of lead inhibition of ferrochelatase; and (3) accumulation of ALA, secondary to inhibition of ALAD, which may stimulate ALAD synthesis in bone marrow cells (Boudene et al., 1984; Fujita et al., 1982).
ALAD is a polymorphic enzyme with two alleles (ALAD 1 and ALAD 2) and three genotypes: ALAD 1,1, ALAD 1,2, and ALAD 2,2. Higher BLL were observed in individuals with the ALAD 1,2 and ALAD 2,2 genotypes compared to similarly exposed individuals with the ALAD 1,1 genotype (Astrin et al., 1987; Hsieh et al., 2000; Wetmur et al., 1991). This observation has prompted the suggestion that the ALAD-2 allele may have a higher binding affinity for lead than the ALAD 1 allele (Bergdahl et al., 1997), a difference that could alter lead mediated outcomes.
Lead in Blood Plasma:
It has been proposed that lead in plasma exists in four states: loosely bound to serum albumin or other proteins with relatively low affinity for lead, complexed to low molecular weight ligands such as amino acids and carboxylic acids, tightly bound to a circulating metallo-protein, and as free Pb2+ (Al-Modhefer et al., 1991). Approximately 40–75% of lead in the plasma is bound to plasma proteins, of which albumin appears to be the dominant ligand. Lead in serum that is not bound to protein exists largely as complexes with low molecular weight sulfhydryl compounds (e.g., cysteine, homocysteine). Other potential lead binding ligands in serum may include citrate, cysteamine, ergothioneine, glutathione, histidine, oxylate and γ-globulins (Al-Modhefer et al., 1991; Ong and Lee, 1980).
Biomarkers of Lead Toxicity:
Measuring blood lead level (BLL) is the most commonly accepted and verifiable biomarker for lead exposure. Lead is unique as a toxicant in that there is agreement among the Centre for Disease Control (CDC), the Agency for Toxic Substances and Disease Registry (ATSDR), and the Environmental Protection Agency (EPA) that there is no toxic threshold for lead (Rousseau et al., 2005; Vorvolakos et al., 2016). This means there is no measurable level of lead in the body below which no harm occurs. The EPA has listed a Maximum Contaminant Level Goal (MCLG) of zero ppb for lead (ATSDR, 2007; Pontius, 1993).
Interference with heme production and subsequent reduction of the heme body pool is one of the main causes of lead related pathology. When whole blood lead levels (BLL) exceed 20 µg/dL the activity of ALAD is inhibited by 50 percent (Jangid, 2012). However, ALAD activity may also be impaired in porphyria, liver cirrhosis and alcoholism. A marked increase in urinary excretion of aminolevulinic acid (ALA), the substrate that accumulates as a result of decreased ALAD, has been used in the past as a marker for lead toxicity, but can be detected only when BLL exceed 35 µg/dL in adults and 25-75 µg/dL in children (Patrick, 2006). Moreover, during heme biosynthesis Lead preclude incorporation of iron into protoporphyrin by inhibiting ferrochelatase activity. Hence, zinc bind with protoporphyrin and form zinc protoporphyrin (ZPP) (Lilis et al., 1978). The presence of ZPP has been proposed as an indicator of recent lead intoxication and thus can be used as a biomarker of exposure (Onalaja and Claudio, 2000).
Worldwide, lead poisoning is an important public health problem, and accounts for nearly 1% of the global burden of disease. The toxic nature of lead was realized more than four thousand years ago (Mathee et al., 2003). However, over the past century in particular, increasingly sophisticated epidemiological studies have more adequately revealed the wide range of health effects that can result from exposure to lead, and that lead can cause health effects at different blood lead levels previously thought to be safe (Vorvolakos et al., 2016). Lead is known to induce a broad range of physiological, biochemical and behavioral dysfunctions in laboratory animals and humans, including central and peripheral nervous systems, haemopoietic system, cardiovascular system, kidneys, liver as well as male and female reproductive systems from many years (ATSDR, 2007, Hernberg, 2000).
Acute, high dose exposure to lead can cause a variety of symptoms, including nausea, vomiting, abdominal pain, malaise, drowsiness, anemia, headaches, irritability, lethargy, convulsions, muscle weakness, ataxia, tremors, paralysis, coma and death (ATSDR, 2007). However, lead has also been shown to affect health in children at very low levels in blood. Chronic, low-level exposure to lead is associated with lowered intelligence quotients, attention deficit disorder and aggression (Mathee et al., 2003).
Workers exposed to lead in manufacturing facilities demonstrate an increased frequency of still births, miscarriages, and spontaneous abortion, reduced sperm counts and motility, decreased fertility, hypospermia, increased rates of teratospermia, and decreased libido. Women who have lead exposed male partners have higher rates of miscarriage. Children of lead exposed workers have increased rates of congenital epilepsy and cardiovascular disease (ATSDR, 2007; Patrick, 2006). In the adult population in general, lead has been associated with hypertension, renal damage and cardiovascular disease (Mir, 2007; Navas-Acien et al., 2007).
At relatively low levels of exposure, adverse health effects may include interference with red blood cell chemistry, delays in normal physical and mental development in babies and young children, slight deficits in attention span, hearing, and learning abilities in children, and slight increases in the blood pressure of some adults (Patrick, 2006).
It appears that some of these effects, particularly changes in the levels of certain blood enzymes and in aspects of children’s neurobehavioral development. The toxic effects of lead vary greatly, manifesting as subtle changes in neurocognitive function in low level exposure or as the potentially fatal encephalopathy of acute lead poisoning. As exposure progresses, symptoms of toxicity may manifest differently (Patrick, 2006). Lead exposure has been related to increased incidence of overall cancers, as well as stomach, lung, and bladder cancer (IARC, 2006).
Lead is capable of interfering with multiple enzymes involved in the production of hemoglobin incorporated into the red blood cell. It has also been found to be capable of eliciting a positive response in a wide range of biological and biochemical tests, which include tests for enzyme inhibition, ﬁdelity of DNA synthesis, mutation, chromosome aberrations, cancer and birth defects. The International Agency for Research on Cancer (IARC) classiﬁed lead as possible human carcinogen (group 2B) and inorganic lead compounds as probable human carcinogens (group 2A) (IARC, 2006). There are several proposed mechanisms to better understand the carcinogenic properties of lead and the conditions required for this purpose. These mechanisms include mitogenesis, alterations in gene transcription, oxidative damage and several indirect genotoxicity mechanisms (García-Lestón, 2010).
Lead Interactions in Heme Biosynthetic Pathway:
The hematopoietic system is one of the target organs in lead toxicity. The enzymes in the biosynthetic pathway of heme in which the effects of lead are of the clinical interest are delta-aminolevulinic acid synthetase (delta-ALAS), delta-aminolevulinic acid dehydratase (delta-ALAD), and ferrochelatase (Carocci et al., 2016). The series of reactions leading to heme biosynthesis begins with succinyl coenzyme A (CoA) and glycine and ends with the insertion of an iron (Fe++) into a molecule of protoporphyrin to form heme. In the first step, the enzyme delta-ALAS catalyzes the formation of delta-ALA from glycine and succinyl CoA, whereas in the second step, delta-ALAD catalyzes the formation of porphobilinogen (PBG) from two molecules of delta-ALA. Due to its affinity for –SH group, lead is known to inhibit delta-ALAD activity that has been used as a laboratory tool for the detection of lead intoxication (Ahamed and Siddiqui, 2007). Over 99% of the lead present in the blood accumulates in erythrocytes. Of this, over 80% is bound to delta-ALAD. Astrin and co-workers (1987) found 50% inhibition of delta-ALAD activity at a BLL of 15 μg/dL. Sakai and Morita (1996) found that threshold value of blood lead for delta-ALAD inhibition was extremely low (approximately 5 μg/dL). In the last step, ferrochelatase incorporates iron (Fe++) into the protoporphyrin molecule to form heme. Lead inhibits ferrochelatase activity and therefore, prevents incorporation of iron into protoporphyrin. This reaction leads to binding of zinc, producing zinc protoporphyrin (ZPP) (Ahamed and Siddiqui, 2007).
Oxidative Stress and Free Radical Toxicity by Lead:
Lead induces the production of reactive oxygen species (ROS) that result in lipid peroxidation, DNA damage, and depletion of cell antioxidant defense systems (Wang et al., 2013). Lead exposure and the resultant oxidative stress have been reported to modify the protein moiety of antioxidant enzymes (Farmand et al., 2005). Oxidative stress appears to be a possible mode of the molecular mechanism of lead toxicity (Wang et al., 2013). Oxidative stress occurs when generation of free radicals (i.e. substances with one or more unpaired electrons) exceed the capacity of antioxidant defense mechanisms (i.e. pathways that provide protection against harmful effect of free radicals). The depletion of glutathione and protein bound sulfhydryl groups and the changes in the activity of various antioxidant enzymes indicative of lipid peroxidation have been implicated in lead induced oxidative tissue damage (Birben et al., 2012). Lead seems to be quite capable of causing oxidative damage to heart, liver, kidney, reproductive organs, brain, and erythrocytes (Ahamed and Siddiqui, 2007). The participation of free radicals in lead toxicity may occur at different levels: (i) inhibition of delta-ALAD by lead accounts for the accumulation of its substrate delta-ALA, that can be rapidly oxidized to generate free radicals as superoxide ion (O2−), hydroxyl radical (OH–), and hydrogen peroxide (H2O2), and (ii) lead has the capacity to stimulate ferrous ion initiated membrane lipid peroxidation (Ahamed and Siddiqui, 2007; Carocci et al., 2016).
Several antioxidant molecules such as glutathione (GSH) and glutathione disulfide (GSSG) levels and antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-s-transferase (GST) and glutathione reductase (GR) activities are the most commonly used parameters to evaluate oxidative damage (Nariya et al., 2017). Glutathione is a tripeptide containing cysteine that has a reactive –SH group with reductive potency. Accordingly, GSH plays a vital role in the protection of cells against oxidative stress. It can act as a non-enzymatic antioxidant by direct interaction of the –SH group with ROS, or it can be involved in the enzymatic detoxification reactions for ROS, as a cofactor or a coenzyme. It possesses carboxylic acid groups, an amino group, a –SH group, and two peptide linkages as sites for reactions of metals. Lead binds exclusively to the –SH group, which decreases the GSH levels and can interfere with the antioxidant activity of GSH (Mabrouk and Ben, 2015). Another component of antioxidant defence system, GR, reduces GSSG back to GSH and thereby supports the antioxidant defence system indirectly. Glutathione reductase possesses a disulfide at its active site that was suggested as target for lead, resulting in the inhibition of the enzyme. This inhibition leads to decreased GSH/GSSG ratios that will render cells more susceptible to oxidative damage. GPx, CAT, and SOD are metalloproteins and accomplish their antioxidant functions by enzymatically detoxifying the peroxides (–OOH), H2O2, and O2. Catalase decomposes H2O2 into H2O and O2–. Glutathione peroxidase needs GSH to decompose H2O2 or other peroxides with the simultaneous oxidation of GSH into GSSG. Catalase has been suggested to provide important pathway for H2O2 decomposition at higher steady state H2O2 concentration, whereas GPx is believed to play a more important role in H2O2 decomposition under lower steady state levels of H2O2 (Carocci, 2016; Ahamed and Siddiqui, 2007). Since these antioxidant enzymes depend on various essential trace elements and prosthetic groups for proper molecular structure and enzymatic activity, they are potential targets for lead toxicity (Kalia et al., 2013). As shown by Othman and El-Missiry (1998), administration of selenium prior to injection of lead into male rats, produced noticeable prophylactic action against lead by means of increased activities of SOD and GR as well as content of GSH. Although the protective effect was attributed to the formation of inactive selenium–lead complex, it was mentioned that such interactions could not be the sole mechanism for the beneficial effects of selenium. SOD dismutates the O2 in to H2O2 and requires copper and zinc for its activity. Copper ions appear to have a functional role in the reaction by undergoing alternate oxidation and reduction, where zinc ions seem to stabilize the enzyme instead of having a role in the catalytic cycle (Gurer and Ercal, 2000). Mylroie and co-workers (1986) observed: (i) high correlation between decreased SOD and decreased copper concentrations in the blood of animals, (ii) no effect on SOD with increased BLLs in the presence of normal copper concentration, and (iii) that dietary copper supplementation prevented the lead induced decrease in SOD activity. Therefore, they have suggested an indirect inhibitory effect on SOD in vivo due to the lead induced copper deficiency. Inhibition of SOD activity by lead was also shown in an in vitro study where the authors indicated that effect of lead can lead to decreased scavenging of ROS and result in oxidative damage (Yun et al., 2011). However, Ariza et al. (1998) demonstrated rapid induction of cellular H2O2 following treatment of AS52 cells with 1M lead, which they suggested to be increased by the stimulatory effect of lead on the activities of CuZn–SOD and xanthine oxidase that produce H2O2. Ribarov and co-workers (1981) found that during Hb oxidation in the presence of lead, H2O2 is generated, which may induce lipid peroxidation in erythrocyte cell membranes. They also found that lead significantly enhances the auto-oxidation of Hb in an in vitro liposome model. The inhibition of this effect by SOD and CAT suggested that O2 and H2O2 are somehow involved in the process. As a result, they speculated that lead might induce generation of ROS by interacting with oxy-Hb, leading to peroxidative damage of erythrocyte membranes (Ribarov et al., 1981).
Genotoxicity of Lead:
A number of studies in different biological systems have used several end points to evaluate the genotoxic effects of lead. The most representative ones are the study of DNA lesions such as structural and numerical chromosome aberration (CA), sister chromatid exchanges (SCEs), micronucleus (MN) test, are more used due to its sensitivity and simplicity (García-Lestón et al., 2010).
Studies showed a significant increase in CA in peripheral lymphocytes of lead workers with high (>30 μg/dL) BLL (Huang et al., 1988; Al-Hakkak et al., 1986: Nordenson et al., 1982; Forni et al., 1976). A significant increase in SCEs was reported by many researchers in lead workers whose BLL was high (>30 μg/dL) (Wu et al., 2002; Duydu et al., 2001; Huang et al., 1988). Grandjean et al. (1983) observed that BLL and SCE rates decreased in lead workers after removal of lead exposure for few days. They also noticed that newly employed workers failed to show any increase in SCE rates during the first 4 months of employment despite increases in both ZPP and BLL, suggesting that genotoxic effects may occur after long exposure to lead. This could also suggest that current BLL is not a good biomarker of genotoxic effects. In contrast, in a group of 18 workers with a mean BLL of 49 μg/dL, there was no detectable increase in SCE frequency relative to controls (BLL <10 μg/dL) (Mäki-Paakkanen et al., 1981). A study of 19 children living in a widely contaminated area reported no significant differences in SCE rates between the exposed children (BLL, 30–60 μg/dL) and 12 controls (BLL, 10–21 μg/dL) (Dalpra et al., 1983).
An increased incidence of micronuclei in peripheral lymphocytes was observed in lead workers as compared to control group of individuals. After the workers consumed a polyvitamin rich diet for 4 months, the micronuclei frequency showed a significant reduction, which led the authors to suggest that oxidative damage might be involved in the genotoxicity of lead (Vaglenov et al., 1998; 2001).
Mechanisms by which inorganic lead is excreted in urine have not been fully characterized. Such studies have been hampered by the difficulties associated with measuring ultra-filterable lead in plasma and thereby in measuring the rate of glomerular filtration of lead. Renal plasma clearance was approximately 20–30 mL/minute in a subject who received a single intravenous injection of a 203Pb chloride tracer (ATSDR, 2007). Measurement of the renal clearance of ultra-filterable lead in plasma indicates that, lead undergoes glomerular filtration and net tubular reabsorption (Araki et al., 1986; 1990; Victery et al., 1979). Renal clearance of blood lead increases with increasing blood lead concentrations above 25 μg/dL (Chamberlain, 1983). The mechanism for this has not been elucidated and could involve a shift in the distribution of lead in blood towards a fraction having a higher glomerular filtration rate (e.g., lower molecular weight complex), a capacity-limited mechanism in the tubular reabsorption of lead, or the effects of lead induced nephrotoxicity on lead reabsorption. Studies conducted in preparations of mammalian small intestine support the existence of saturable and non-saturable pathways of lead transfer and suggest that lead can interact with transport mechanisms for calcium and iron (ATSDR, 2007).
In humans, absorbed inorganic lead is excreted in feces (ATSDR, 2007). The mechanisms for fecal excretion of absorbed lead have not been elucidated; however, pathways of excretion may include secretion into the bile, gastric fluid and saliva (Rabinowitz et al., 1976). Biliary excretion of lead has also been observed in the dog, rat, and rabbit (Klaassen and Shoeman, 1974).
The use of plant and plant products for treatment of adverse effects is as old as mankind. The major advantages of plant based remedy having efficacy, cheap, easily available and no incidences of serious adverse effects. Several studies revealed that experimental research has been escalated in pursuit of medicinal plants and natural products that could abrogate metal toxicity in animals and humans. In this study, the toxicity of lead was tried to mitigate by curcumin and S-Allylcysteine – a sulfur compound of garlic. Hence, brief introduction of both given here.
Curcumin is a substance obtained from plant Curcuma longa L. The origin of the plant Curcuma longa L., which belongs to Zingiberaceae family is India. (Velayudhan et al., 2012). The plant is distributed throughout tropical and subtropical regions of the world, being widely cultivated in Southeast Asian countries. These powdered dried rhizomes have at least 76 synonyms listed in the 1999 WHO monograph (WHO, 1999). Among them some important names are Haldi (in Hindi), Haridra or Gauri (in Sanskrit), Chiang Huang (in Chinese), Ukon (in Japanese), Kurkum (in Arabic), Besar (in Nepali) etc. It is measures up to 1 m high with a short stem and tufted leaves. Turmeric, i.e., the ground rhizome of Curcuma Longa L., has a long history of use in food as a spice, mainly as an ingredient in many verities of curry powders and sauces, where curcumin from turmeric is a main colouring substance, imparting colour and flavour to the food. Most active component in turmeric is curcumin, which may make up to 2-5 % of the total spice in turmeric (WHO, 1999).
It is the major constituent of the oleoresin of turmeric in the crude extract of rhizomes of Curcuma longa about 70-76% curcumin is present along with about 16% demethoxycurcumin and 8% bismethoxycurcumin (Kohli et al., 2005). It is extensively used in the traditional Indian medicine to treat a wide variety of diseases. The curcuminoids, demethoxycurcumin and bisdemethoxycurcumin are polyphenols and are responsible for the yellow colour of turmeric. They are deriving from turmeric by ethanol extraction. The pure orange yellow crystalline powder is insoluble in water. Curcumin can exist in several tautomeric forms including keto and enol forms. The enol form is more energetically stable in the solid phase and in solution (Patel et al., 2014). The sequencing in which the function groups, the alcohol and the methoxy, introduced themselves onto the curcuminoid seems to involved two cinnamate units being coupled together by malonyl-CoA. (Kita et al., 2008; Roughley and Whiting, 1973). It utilizes cinnamic acid as their starting point, which is derived from the amino acid phenylalnine. This is noteworthy because plant biosynthesis employing cinnamic acid as a starting point are rare compared to the more common use of p-coumaric acid (Kita et al., 2008).
Metabolism and Metabolites of Curcumin:
There are two main transformation pathways of curcumin, namely, reduction and glucuronidation. NADPH is required for reduction reaction but the nature of the reductase is unknown. Furthermore, most of the conjugated derivatives are hydrolyzed by β-glucuronides. The experimental results suggested that curcumin was first biotransformed into dihydrocurcumin and tetrahydrocurcumin and these compounds subsequently were converted to monoglucuronides conjugates. Thus, it was suggested that curcumin- glucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are major metabolites of curcumin in vivo (Pan et al., 1999).
Potential Uses and Protective effects of Curcumin:
India produces most of the World’s supply of turmeric (Khan and Abourashed, 2011). It has been used by the food industry as additive, flavouring, preservative, and colouring agent (e.g., in mustard, margarine, soft drinks and beverages). As a safe colouring agent, curcumin is listed in the international numbering system for food additives with the code E100 (Strimpakos and Sharma, 2008). Non-medical human application of turmeric includes cosmetics which contain essential oils, polyphenols, protein, fat, minerals, carbohydrates and moisture. The aromatic properties of turmeric are thought to be attributable to its volatile essential oils (Burt, 2004).
In an Indian Ayurvedic medicine turmeric has been used since 1900 BC to treat a wide variety of ailments. Research in the latter half of the 20th century has identified curcumin as responsible for most of the biological activity of turmeric (WHO, 1999). As a traditional medicine, turmeric has also been extensively used for centuries to treat a diversity of disorders including rheumatism, body ache, hepatic disorders, biliousness, urinary discharges, dyspepsia, inflammation, constipation, leukoderma, amenorrhoea and colic inflammation. Numerous clinical trials in human were under ways, studying the effect of curcumin in numerous diseases including multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and Alzheimer’s disease (Goel et al., 2008; Gupta et al., 2013; Sanmukhani et al., 2014).
In vitro and animal studies have also suggested that curcumin may have antitumor, antioxidant, antiarthritic, anti-amyloid, anti-ischemic and many other protective effects along with anti-inflammatory properties (Aggarwal et al., 2007; Zorofchian et al., 2014). In addition, it may be effective in treating malaria, prevention of cervical cancer, hepatoprotective and may interfere with the replication of the HIV virus (Jacob, 2011). Study showed that curcumin inhibited the recruitment of RNA polymerase II to viral DNA, thus inhibiting the transcription of the viral DNA (Kutluay et al., 2008). Curcumin is also significantly associated with protection from infection by HSV-2 in animal models of intravaginal infections (Zorofchian et al., 2014). Curcumin act as a free radical scavenger and antioxidant by inhibiting lipid peroxidation (Jagetia and Rajanikant, 2015). A study involving genetically altered mice suggest that curcumin might inhibit the accumulation of destructive beta-amyloid in the brains of Alzheimer’s diseases patient and also break up exiting plaques associated with the disease (Mishra and Palanivelu, 2008). Its potential anticancer effects seen from its ability to induce apoptosis in cancer cells without cytotoxic effects on healthy cells. Because the gastrointestinal tract seems to be exposed more prominently to unmetabolized curcumin than any other tissue, the results support the clinical evaluation of curcumin as a colorectal cancer chemopreventive agent (Bagchi and Preuss, 2004). Curcumin can interfere with the activity of the transcription factor NF-ҡB, which has been linked to a number of inflammatory diseases such as cancer (Sikora et al., 2010). Curcumin and its complex with manganese offer protective action against neurological disorder like dementia by exerting antioxidant activity (Thiyagarajan and Sharma, 2004). Role of curcumin on metal induced toxicity was also documented (Oguzturk et al., 2012; Rao et al., 2008). Recently, Nguyen et al. (2017) reported that the ether, alcohol and water extract of C. longa show anti-inflammatory effects.
The antioxidant activity of curcumin was identified very long back (Sharma, 1976). It acts as a scavenger of oxygen free radicals. In vitro, curcumin can significantly inhibit the generation of reactive oxygen species (ROS) like superoxide anions, H2O2 and nitrite radicals generated by activated macrophages, which play an important role in inflammation. Curcumin also lowers the production of ROS in vivo (Chattopadhyay et al., 2004). It can protect hemoglobin from oxidation. Its derivatives, demethoxycurcumin and bisdemethoxycurcumin also have antioxidative effects (Howes and Houghton, 2009). Curcumin is a potent scavenger of a variety of ROS, including superoxide anion, hydroxyl radical, singlet oxygen, nitric oxide and peroxynitrite (Ak and Gülçin, 2008). Curcumin has the ability to protect lipids, hemoglobin and DNA scavenging activity than demethoxycurcumin or bisdemethoxycurcumin. Curcumin is also a potent inhibitor of ROS-generating enzyme cyclooxygenase and lipoxygenase in mouse epidermis (Aggrawal, 2007).
Mechanism of Action:
Curcumin shows its antioxidant activity due to its unique conjugated structure, which includes two methoxylated phenols and enol form of β-diketone; the structure shows typical radicals trapping ability as a chain-breaking antioxidant (Chattopadhyay et al., 2004). Generally, the non-enzyme antioxidant process of the phenolic materials is thought to be mediated through the two stages: Radical trapping stage and Radical termination stage (Valgimigli and Pratt, 2015).
Bioavailability of Curcumin:
Numerous studies have been performed on the biotransformation of curcumin. Dissolving curcumin in hot water prior to ingestion, or in warm oily liquids, appears to increase bioavailability. Researcher has developed a number of curcumin analogs that appear to have greater bioavailability, but these analogs have not been tested broad, either in vitro or in vivo, for medicinal purposes (Prasad et al., 2014). Aggarwal and co-workers (2014) showed that curcumin was first biotransformed to dihydrocurcumin (DHC) and tetrahydrocurcumin conjugates. Curcumin-glucurnoids, dihydro-curcuminglucurnoide, tetrahydrocurcumin-glucurnoide and tetrahydrocurcumn are major metabolites of curcumin in mice. Shoba et al. (1998) found that curcumin has poor bioavailability due to its rapid metabolism in the liver and intestinal walls. In their study, the effects of combining piperine, a known inhibitor of hepatic and intestinal glucurnoidation, was evaluated on the bioavailability of curcumin in rats and healthy human volunteers. The study showed that in the dosages used, piperine enhances the serum concentration, extent of absorption and bioavailability of curcumin in both rats and humans, with no adverse effects. A polymeric nanoparticle encapsulated formulation of curcumin, “nanocurcumin” has been also synthesized. It has the potential to bypass many of the shortcomings associated with free curcumin, such as poor solubility and poor systemic bioavailability. Nanocurcumin particles have a size of less than 100 nanometers on average, and demonstrate comparable to superior efficacy compared to free curcumin in human cancer cell line models (Bisht et al., 2007).
S-ALLYLCYSTEINE (SAC) – A SULFUR COMPOUND OF GARLIC
The genus Allium includes garlic, scallions, onions, chives, and leeks. These all contain the sulfur compounds which are medicinally active. A member of the Liliaceae family, garlic (Allium sativum) is a cultivated plant highly regarded throughout the world. Originally from Central Asia, garlic is one of the earliest of cultivated food. Garlic is a versatile vegetable often used as ingredient in many dishes for flavour, aroma and taste enhancement. It has been also used as an effective remedy for a variety of ailments (Cardelle-cobas et al., 2010). Most often it is used raw after being chopped, minced, sliced, or juiced. Many times, it is cooked also where it enhances flavour as well as adds nutritional benefit. Different garlic dietary supplements including dried or powdered formulations, oils and liquid extracts have been incorporated into the market to satisfy the demand of consumer for garlic bio active compounds. Among garlic derived products, Aged garlic extract (AGE) is the only preparation with the highest antioxidant activity, even more than fresh garlic and other commercial garlic supplements (Colín-González et al., 2012; Freeman and Kodera, 1995; Imai et al., 1994).
Early men of medicine such as Hippocrates, Pliny and Aristotle promoted a number of therapeutic uses for this plant (Murray et al., 2005). In addition to its reputation as a healthy food, several studies had shown garlic as anti-viral, anti-bacterial, anti-fungal and antioxidant capacities. Additionally, anti-atherosclerotic and anti-cancer properties have also been demonstrated. Garlic and its various preparations may offer simple remedies for ailments from common cough and colds to whooping cough and other pulmonary diseases, skin troubles, gastrointestinal disorders, for averting premature ageing and for improving immunity (Bongiorno et al., 2008; Lanzotti, 2006; Das, 2002). During World War I, garlic poultices were used topically to prevent wound infections. By World War II, garlic had a reputation as “Russian penicillin” (Cardelle-Cobas et al., 2010; Kemper, 2000).
Garlic bulbs are the source of many compounds with medicinal properties with varied pharmacological functions thus supporting many of the traditional medicinal uses of garlic. The major constituents of garlic are alliin, S-Allylcysteine, ajoene, diallylsulfide, diallyldisulfide, diallyltrisulfide, 2-vinyl-4H1,2 dithin, linoleic acid, scordinins and selenium (Omar and Al-Wabel, 2010; WHO, 1999). However, despite the fact that the above-mentioned compound contributes in part to garlic bioactivity, evidence from several investigations suggests that the biological and medical functions of garlic are mainly due to their high content in organo-sulphur compounds, which likely work synergistically with other compounds such as organo-selenium compounds. Despite the numerous therapeutic effects attributed to garlic, the chemistry behind it’s health promoting effects is still poorly understood.
Medical Uses of Garlic:
Garlic has attracted much attention, since the National Cancer Institute in the USA selected garlic for its Designer Food Program for the research of cancer preventive materials. With regard to the constituents of garlic, there are many reports about the chemical analysis and biological activities of sulphur containing compounds of raw garlic and their transformed products in several different types of preparations (Lawson, 1993; Kemper, 2000; Nicastro et al., 2015).
Garlic’s current principal medicinal uses are to prevent and treat cardiovascular disease by lowering blood pressure, as an antimicrobial, and as a preventive agent for cancer. Garlic lowers total cholesterol concentrations by approximately 10% and favorably alters HDL/LDL ratios. Garlic also inhibits platelet aggregation and enhances fibrinolytic activity, reducing clots on damaged endothelium (Chaturvedi and Chaturvedi, 2011; Londhe, 2014). It is a good source of dietary phytochemicals with proven antioxidant properties and ability to modulate the detoxification systems (Nuutila et al., 2003). Researchers isolated and identified several flavonoids and sulphur-containing compounds (diallyl sulphide, trisulphide and allyl-cystein) in garlic (Kodera et al., 2002). These are likely to play an important role in the widely demonstrated biological effects of garlic, which include anti-tumour (Ejaz et al., 2003), hypolipidemic, hypocholesterolemic, antiatherosclerotic, antioxidant (Chowdhury et al., 2008) and immunomodulatory (Bruck et al., 2005) effects.
S-Allylcysteine (SAC), a Sulfur Compound of Garlic:
SAC is one of the water-soluble, sulfur containing, transformational product from garlic derived from γ-glutamyl peptides. SAC is contended to be the only compliance marker compound for clinical studies involving garlic consumption. In addition to odoriferous oil soluble compounds, less odorous, water soluble, organosulphur compounds such as SAC and S-allylmercacyeteine have shown to be biologically active in several areas. The non-volatile sulphur containing compounds SAC are present in several garlic preparations, although the content varies considerably (Cardelle-Cobas, 2010; Imai et al., 1994; Lawson, 1993). Nagae et al. (1994) found that SAC was rapidly and easily absorbed in the gastrointestinal tract and, then distributed mainly in the plasma, liver and kidney after oral consumption.
Antioxidant Properties of SAC:
Free radicals which include reactive oxygen species (ROS), are toxic by-products of a normal metabolism. ROS increase during inflammation, exercise and exposure to environmental pollutants, radiation and sunlight. ROS damage DNA, lipids and proteins which leads to aging and disease. Aged garlic extract’s antioxidant activity enhances the body’s ability to make glutathione and providing powerful protection against ROS damage (Colín-González et al., 2012).
Antioxidant properties have been documented for garlic in vitro and in vivo studies (Ebadi, 2006). Garlic constituents inhibit the formation of free radicals, support endogenous radical scavenging mechanisms, enhance cellular antioxidant enzymes, protect low-density lipoprotein from oxidation by free radicals and inhibit the activation of the oxidant-induced transcription factor NF-ҡB (Borek, 2001). Many previous investigations demonstrated that garlic organo-sulphur compounds prevent toxicity induced by cyanide, sodium nitrite (Helal and Elsaid, 2006), carbon tetrachloride (Kodai et al., 2007), lead (Kianoush et al., 2012), chromium (Kalayarasan et al., 2008) and ethanol (Hussein, 2003). The toxicity mechanism for all of them entails oxidative stress and impairment in the antioxidant defence systems (Asadpour et al., 2013).
SAC have been shown to exert antioxidant effects through their ability to (1) scavenge reactive oxygen species, (2) inhibit lipid peroxidation, (3) reduce ischemic/reperfusion damage, (4) inhibit oxidative modiﬁcation of low density lipoproteins and (5) prevent oxidative damages to DNA (Colín-González et al., 2012; Awazu and Horie, 2008; Borek, 2001). SAC may act synergistic with other antioxidants in an additive way. In addition to scavenging ROS, they exert their antioxidant action by enhancing the activity of the cellular antioxidant enzymes superoxide dismutase, catalase and glutathione peroxide, and increasing the glutathione in cells (Colín-González et al., 2012). This is an important defense mechanism in living cells, since, in addition to protecting against oxidative stress and being a cofactor for the antioxidant enzyme glutathione peroxidase is one of the detoxification system of the body and induces the detoxifying enzymes. Thus, they provide additional protection to own antioxidant defenses of organism against oxidative damage, decreasing the risk of injury to vital molecules and helping to prevent the onset and progression of diseases (Colín-González et al, 2012; Borek, 2001).
SAC demonstrated a scavenging effect on hydrogen peroxide and also inhibited the chain oxidation induced by a hydrophilic radical initiator (Ide et al., 1996). Five cysteine containing compounds derived from garlic, including SAC, were able to enhance GSH levels and Catalase as well as GPx activity in the kidney and liver (Hsu et al., 2004). The cysteine containing compounds were also found to decrease fibronectin, triglyceride, and cholesterol levels in plasma and liver, while they increased levels of α-tocopherol in the liver, plasma, and kidney (Hsu et al., 2004). Padmanabhan and Prince (2006) also concluded improvements in lipid peroxide markers and antioxidant status due to the antioxidative effects of SAC. It significantly inhibited nuclear damage caused by dimethyl hydrazine, thus decreasing the toxicity of this carcinogen by stimulation of the activity of glutathione S-transferase in both the liver and colon (Pandrangi, 2015). SAC has been shown to protect hepatocytes (Kalayarasan et al., 2008), a chemopreventive effect on colon cancer (Hatono et al., 1996) and an antiproliferative effect on human neuroblastoma cells (Welch et al., 1992).
In the light of aforementioned beneficial properties of curcumin from Turmeric and SAC from Garlic, this study was carried out to investigate the possible in vitro protective effects of this agents on lead induced genotoxicity, free radical toxicity and other biochemical impairment in blood sample of human.