Abstract
Lead (Pb) toxicity is a public health problem affecting millions worldwide. Advances in ‘omic’ technology have paved the way to toxico-genomics which is currently revolutionizing the understanding of interindividual variations in susceptibility to Pb toxicity and its functional consequences to exposure. Our objective was to identify, comprehensively analyze, and curate all the potential genetic and epigenetic biomarkers studied to date in relation to Pb toxicity and its association with diseases. We screened a volume of research articles that focused on Pb toxicity and its association with genetic and epigenetic signatures in the perspective of occupational and environmental Pb exposure. Due to wide variations in population size, ethnicity, age-groups, and source of exposure in different studies, researchers continue to be skeptical on the topic of the influence of genetic variations in Pb toxicity. However, surface knowledge of the underlying genetic factors will aid in elucidating the mechanism of action of Pb. Moreover, in recent years, the application of epigenetics in Pb toxicity has become a promising area in toxicology to understand the influence of epigenetic mechanisms such as DNA methylation, chromatin remodeling, and small RNAs for the regulation of genes in response to Pb exposure during early life. Growing evidences of ecogenetic understanding (both genetic and epigenetic processes) in a dose-dependent manner may help uncover the mechanism of action of Pb and in the identification of susceptible groups. Such studies will further help in refining uncertainty factors and in addressing risk assessment of Pb poisoning.
Introduction
Lead (Pb) is one of the earth’s oldest dynamic metals and a potent environmental, nonbiodegradable, public health toxin. For decades, this wonder metal has made a great impact on the growth of the world economy. It has been roughly estimated that about 90% of mined Pb is consumed into the industrial sector for various applications such as in the manufacture of acid storage batteries, production of various metal alloys, for its use in ammunition, construction materials like Pb sheets, pipes, solders, in few brass and bronze products, in preparation of oil and paints, cosmetics, printing ink, herbal medicines, cultural powders, and several others, exposing the general population including pregnant woman and children. 1 –3 Pb poisoning accounts for about 0.6% of the global burden of diseases. 4 Several efforts to limit the application of Pb in daily products are ongoing but it continues to remain a silent threat to the general population. The US Centers for Disease Control and Prevention designated a blood Pb reference value of 5 µg/dL in children and 10 µg/dL in adults. 5,6 The National Institute for Occupational Safety and Health which is also a part of the US Centers for Disease Control and Prevention has designated a blood Pb reference value of 5 µg/dL for occupationally exposed adults. 7 However, cases of adult and childhood Pb poisoning continue to be reported due to association of low Pb-B levels with disease processes such as learning and behavioral deficits, 8,9 cardiovascular 10 and renal 11 disease, decreased fertility, 12,13 and cancer 14 suggesting no safe threshold level for Pb. Pb is known to mimic common divalent metal ions such as calcium (Ca), iron (Fe), zinc (Zn), copper (Cu), and magnesium (Mg), hence altering the biological functions. 15 This facilitated the identification of metabolic pathways and gene networks associated with Pb opening doors to understand the toxico-genomics, -epigenomics and -metabolomics of Pb as depicted in Figure 1. Despite the clinical consequences of Pb exposure, the mechanism of Pb entry into target cells and the genes that may be involved in modifying the toxicokinetics and toxicodynamics of Pb is still not clear. 16 Every individual is continually exposed to the sources of Pb either through occupational or environmental means. Interindividual variation to the toxic effect of Pb depends on factors like age, gender, nutrition, ethnicity, lifestyle, amount and duration of Pb exposure. Consequently, risk assessment and decision-making concerning Pb poisoning is hampered and remains uncertain. Knowledge of the origin and nature of Pb in the exposure setting is also crucial in understanding the interindividual variations in the toxicokinetic–toxicodynamic mechanism of Pb which will further enable us to assess cumulative exposure and signs of toxicity in a better way. This review broadly outlines the genetic and epigenetic outcomes associated with Pb poisoning conducted to date.
Molecular pathways and gene networks affected by Pb. (1) Pb2+ triggers the oxidative stress mechanism via generation of ROS resulting in the (a) deterioration of antioxidant pool and increase in antioxidant enzyme activity; (b) oxidation of membrane phospholipids. (2) Pb2+ enters the cell via the Ca channel (stimulated by the G-protein activated signaling) and binds to calmodulin with higher affinity than Ca2+. It further causes the phosphorylation of dependent protein kinases (PKC) leading to abnormal enzyme activity and gene transcription. (3) In an alternative route, the G-protein activates the PLC which in turn activates the hydrolysis of PIP2 to IP3 and DAG. IP3 releases Ca2+ from the endoplasmic reticulum which then binds to calmodulin. In the presence of Pb2+ inside the cell organelle, IP3 causes the release of Pb2+ to bind to calmodulin. (4) Pb2 disrupts the cellular regulation of Fe2+ via hemochromatosis (HFE) protein. (5) Pb2 also enters the cell via the divalent metal transporter (SLC11A2) which is specific to Fe2+. (6) In response to heavy metal stress, the cell expresses MTs which is essential for restoring metal homeostasis and in detoxification processes. (7) Chronic exposure to Pb2 may affect mitochondrial Ca2+ regulation triggering bioenergetic failure through the opening of permeability transition pore, release of cytochrome c and other proapoptotic factors leading to cellular death by apoptosis and necrosis. (8) Pb2 inhibits cytosolic enzyme, ALAD and blocks heme synthesis. (9) Pb2 inhibits Na+ K+ ATPase by replacing Na+, in turn affecting the normal functioning of a cell. (10) Nanomolar concentrations of Pb2 (or low Pb exposure) can activate important transcription factors that are involved in cellular proliferation, apoptosis, host–defense mechanism and inflammation. Pb: lead; ROS: reactive oxygen species; Ca: calcium; PKC: protein kinase C; PLC: phospholipase-C; PIP2: phosphatidylinositol 4,5 bisphosphate; MT: metallothionein; ALAD: δ-aminolevulinate dehydratase; IP3: inositol trisphosphate; DAG: diacylglycerol.
Environmental response machinery and genetic factors influencing Pb toxicity
The Rio convention of 1993 identified genetic variations as one of the three pillars of biodiversity in addition to ecosystems/landscapes and species richness. 17 The human body has inbuilt sophisticated pathways to minimize the biological consequences of toxic exposure by developing an environmental response machinery. 18 Since the human genome is subjected to genetic variability, variations in the components of the environmental response machinery may contribute to alterations in the efficiency of biological pathways. These environmentally responsive genes belong to metabolisms that control biotransformation, DNA repair, immunity, cell cycle, and signaling from which biomarker information of Pb exposure, effect, and of susceptibility can be elucidated. Thus, depending on the efficiency of environmental response genes or selectable markers, an individual’s risk of Pb exposure and toxicity can be interpreted. This is an emerging field in human genetics known as Ecogenetics. It is technically defined as the “study of critical genetic determinants that dictate susceptibility to environmentally influenced adverse health effects.” 19,20 This field investigates how genetic variations can act as risk factors for several diseases including cardiovascular, diabetes, cancer, and exposure to xenobiotics. Pb is a slow poison which takes years to manifest its toxic effects. Hence, it becomes difficult to quantify its lethal effects unless it has developed a diseased phenotype. Recently, increasing number of studies has started to document genetic and epigenetic factors that may influence the toxicokinetics and toxicodynamics of Pb in association with disease. 21
Decades of research have identified genes that may induce susceptibility to Pb toxicity such as ALAD (δ-aminolevulinate dehydratase; Gene ID: 210; Accession no.: NC_000009.12), 22 –25 VDR (Vitamin D receptor; Gene ID: 7421; Accession no.: NC_000012.12), 26 –29 G6PD (Glucose-6-phosphate dehydrogenase; Gene ID: 2539; Accession no.: NC_000023.11), HFE (Hemochromatosis; Gene ID: 3077; Accession no.: NC_000006.12), 30 –32 CYP1A2 (Cytochrome P450 Family 1 Subfamily A Member 2; Gene ID:1544; Accession no.: NC_000015.10), CYP2D6 (Cytochrome P450 Family 2 Subfamily D Member 6; Gene ID:1565; Accession no.: NC_000022.11), 33 MGP (Matrix γ-carboxyglutamate; Gene ID:4256; Accession no.: NC_000012.12), 34 SLC11A2 (Solute Carrier Family 11 Member 2; Gene ID: 4891; Accession no.: NC_000012.12), 35 –37 TF (Transferrin; Gene ID:7018; Accession no.: NC_000003.12), 38 MT2A (Metallothionein 2A; Gene ID: 4502; Accession no.: NC_000016.10), 39 DRD4 (Dopamine receptor; Gene ID: 1815; Accession no.: NC_000011.10), 40 and XRCC1 (X-ray repair cross-complementing protein; Gene ID: 7515; Accession no.: NC_000019.10). 41 Table 1 presents a list of single nucleotide polymorphisms that has been studied in association with Pb toxicity so far. Cumulative studies on the impact of genetic predisposition of Pb toxicokinetics however are limited due to wide variations in individual genotype phenotype combinations, source of contamination, and magnitude of exposure. This calls for studies to detect functional gene polymorphisms related to Pb-associated disease phenotypes, the impact of relevance of genetic variations and gene–gene interactions. An understanding of the genetic factors underlying susceptibility to Pb toxicity may have a significant importance in the risk assessment of occupationally exposed and general population leading to individualized or personalized intervention for Pb toxicity. 60
Genes extensively studied for investigating the influence of polymorphisms on Pb toxicity.a
aThe em dash sign indicates no information available.
Genetic association with Pb toxicokinetics
Pb and heme pathway
One of the main systems affected by Pb is the hematopoietic system. Pb is known to inhibit δ-aminolevulinate dehydratase (ALAD), the second enzyme involved in the conversion of two molecules of aminolevulinate to porphobilinogen. Pb has a strong binding capacity for proteins containing sulfhydryl groups, hence interfering with the activity of enzymes and structural proteins. 61 The absorption of Pb can cause Fe deficiency resulting in anemia. In this section, we are focusing on two enzymes, ALAD and Ferrochelatase (FECH) involved in the heme synthesis and a heme protein CYP450 which are potential targets of Pb.
δ-Aminolevulinate dehydratase
ALAD (EC 4.2.1.24) is a cytosolic enzyme which catalyzes the conversion of aminolevulinate to porphobilinogen in the heme pathway. Pb inactivates ALAD by displacing Zn from its active site. The most studied ALAD polymorphism (rs1800435) is the G to C transversion at the 177 position of exon 4. 62,63 ALAD has been one of the earliest genes investigated for its role in genetic susceptibility to Pb poisoning. The polymorphic nature of ALAD results in three phenotypic variants designated as ALAD 1-1, ALAD 1-2, and ALAD 2-2. It has been observed that the ALAD 2 phenotype has higher affinity and stability for Pb compared to ALAD 1 which thereby reduces the bioavailability of Pb for other potential enzymes such as Coproporphyrinogen oxidase (CPOX) and FECH. 23,62 There are ample literatures supporting data that ALAD polymorphism has a role in modifying the toxicokinetics of Pb. Several studies have observed that subjects who had at least one copy of the ALAD 2 allele compared to subjects with absence of allele is associated with various factors such as high Pb-B levels, lower dimercaptosuccinic acid (DMSA)-chelatable Pb levels, lower plasma aminolevulinic acid (ALA-P) levels, a larger difference between trabecular and cortical bone Pb levels, higher blood urea nitrogen (BUN) and serum creatinine (S-Cr) levels, less efficient uptake of Pb into bone, especially trabecular, lower Zn protoporphyrin (ZPP) levels for given levels of Pb-B, and lower urinary calcium (U-Ca) and creatinine (Cr) levels. 64 This comprehensive information prompted a meta-analysis of ALAD rs1800435 polymorphism on Pb-B levels by Scinicariello et al. based on nine occupational and five environmental exposure studies. 65 The analysis revealed that 87.84% of the subjects carried ALAD 1-1 wild type allele while 12.16% were homozygous for mutant ALAD 2-2. The weighted mean difference pooled data analysis indicated that ALAD 2 carriers had significantly higher Pb-B levels compared to ALAD 1 carriers. However, further analysis revealed that at Pb-B concentrations <10 μg/dL, the ALAD 2 allele showed negligible influence on susceptibility. Similarly, another meta-analysis by Zhao et al. showed that ALAD 2 carriers had significantly higher Pb-B levels than ALAD 1-1 homozygotes indicated by the fixed effect and random effect models. 66 Recently, our laboratory published findings that ALAD 2 carriers were susceptible to elevated Pb-B levels compared to ALAD 1 carriers. 63 Further, there has been reports of association of ALAD polymorphism on placental Pb levels. Further, ALAD has been associated with Pb neurotoxicity. Sobin et al. showed that with increase in Pb-B level, the ALAD 2 genotype was associated with enhancement of visual attention and working memory. 42 Yun et al. showed that neonatal Neonatal Behavioral Neurological Assessment (NBNA) score of mothers carrying ALAD 1-2 genotype and with higher Pb-B levels was low compared to neonatal NBNA scores of mothers carrying the ALAD 1-1 genotype indicating that ALAD polymorphism may affect the neonatal NBNA score by influencing Pb-B levels. 43 In association with renal function, it was observed that ALAD 2 carriers had significantly higher S-Cr and BUN levels than ALAD 1 carriers. 64,67 Conversely, there are studies which report the opposite of this which may be due to heterogeneity in the level of exposure and frequency of polymorphism in the population investigated. 68 Exposure to environmental or occupational Pb resulting in Pb-B levels in the range of 10–40 µg/dL may be an independent factor in the manifestation of cardiovascular events in association with arterial hypertension. 69,70 In association with blood pressure, three cross-sectional studies revealed that ALAD 2 carriers environmentally exposed to Pb had increased diastolic blood pressure of 1.8 mm/Hg (p = 0.01) but no significant association with systolic blood pressure. 64,71,72 In addition to this, several other polymorphisms located in exon 4 (Rsa), exon 5 (Rsa39488), intron 6 (HpyIV and HpyCH4), and intron 12 (Sau3A) were investigated for their association with renal functions among Pb-exposed groups. Chia et al. observed that for every 1 µg/dL rise in Pb-B, the HpyCH4 1-1 showed few points fold increase in creatinine (Cr) urinary (U) α1 microglobulin, Cr U β2 microglobulin, Cr U retinol binding protein, and U albumin levels compared to HpyCH4 1-2 variants indicating the resistant role of HpyCH4 1-2 to the effects of Pb toxicity on renal function. 44 Another study showed that the risk of renal carcinoma associated with Pb exposure was highest among subjects with homozygous wild/mutant and heterozygous variant of ALAD rs8177796 and homozygous wild of ALAD rs2761016 while decreased risk was observed in subjects with rs2761016 homozygous variants. 46 Another group studied the effect of three ALAD polymorphisms (rs1805313, rs2228083, and rs1139488) on Pb-B levels of occupationally exposed workers and they observed that the T and C allele of locus rs1805313 (C/T) and rs1139488 (T/C) and the heterozygous variant of rs2228083 (T/C) might determine higher Pb-B levels. 47 These observations infer that ALAD may significantly modify the toxicokinetic and toxicodynamics of Pb. However, as far as genetics goes, the role of ALAD polymorphism in the bioaccumulation of Pb-B is ambiguous due to wide variations in ethnicity, study population size, interindividual metabolic differences, and source of exposure. A more meaningful way to interpret ALAD in Pb toxicity is dose-dependent gene expression studies. Now epigenetic research in toxicology is gaining momentum to understand the quality and expression of several candidate genes in both in vivo and in vitro conditions. The final section is dedicated to the epigenetics of Pb toxicity.
Ferrochelatase
FECH (EC 4.99.1.1) is a mitochondrial enzyme which catalyzes the insertion of ferrous (Fe2+) ion into the protoporphyrin ring forming heme. 73 Deficiency of FECH is marked by the increased accumulation of erythrocytic or ZPP resulting in erythropoietic protoporphyria (EPP). 74 The ZPP levels rise exponentially at Pb-B concentrations >30 μg/dL in adults and >25 μg/dL in children. 75 Besides studies focusing on the association of Fe, ZPP levels, and FECH activity, FECH (Gene ID: 2235; Accession no.: NC_000018.10) mutations in association with EPP and its modifying role with respect to Pb poisoning is lacking. Rogan et al. reported an association of FECH and ALAD activity in Pb-exposed nonanemic children. The meta-analysis by Scinicariello et al. also showed that ALAD 2 isozyme decreased the bioavailability of Pb to inhibit FECH compared to ALAD 1 indicating a modifying effect of ALAD polymorphism on activity of FECH. 65
CYP450 (Cytochrome P450)
Liver is known to actively participate in the detoxification of drugs and xenobiotics. This metabolic clearance is aided by a host of sulfhydryl, heme-containing monooxygenases called cytochrome P450. A remarkable amount of it is found not only in the liver but also in the small intestine. More than 50 isoforms of CYP450 have been identified, of which CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 enzymes are known to metabolize 90% of drugs among which CYP2C19 and CYP2D6 is mostly affected by various genetic changes. 76,77 Animal studies revealed that Pb intoxication reduces the cytochrome dependent P450 microsomal enzyme content and its activity without altering the amount of hepatic CYP450. Degawa et al. reported the inhibition of microsomal CYP1A2 activity and reduced expression of its mRNA and protein in rat liver by Pb. 78 Very few human studies have evaluated the impact of Pb on the activity of cytochrome P450. Saenger et al. suggested the effect of Pb on CYP3A4 activity while Fishbein et al. observed minimal effect on hepatic cytochrome P450 of chronically Pb-intoxicated demolition workers. 79,80 This high inter-subject variability in CYP450 activity is largely attributed by genetic polymorphisms which in turn may influence an individual’s response to a drug or toxic substance. 76 Other studies by Lowry et al. observed no impact of CYP1A2 and CYP2D6 polymorphisms on low Pb-B levels but it was concluded that high Pb concentrations may induce biotransformation of drugs which can act as substrates for CYP1A2 and CYP2D6. 33 Therefore, genetic variations of CYP450 need to be assessed to understand its role in metabolizing Pb. Currently, with so many advances in genomics, this variation can also be explained by epigenetic regulation and regulation by noncoding RNAs in genes controlling the expression of cytochrome.
Pb and oxidative stress
Pb is a redox inactive metal which induces oxidative stress indirectly by depleting the antioxidant reserves of the cell by selectively binding to thiol containing antioxidants and enzymes mainly superoxide dismutase, catalase, and glutathione peroxidase thus generating reactive oxygen species (ROS). 81 Consequently, critical biomolecules such as DNA, proteins, and lipids are damaged. Pb also induces oxidative stress via inhibition of ALAD resulting in the increased accumulation of its substrate aminolevulinic acid (ALA), a potent neurotoxin. 81,82 The extensive production of ROS by Pb reduces the antioxidant, glutathione (GSH), a sulfhydryl antioxidant which represents 90% of nontissue sulfur pool of the human body and associated with quenching of free radicals. 83,84 Although alternative pathways to synthesize GSH from cysteine via the γ-glutamyl cycle exists, it fails to replenish the depleted GSH levels in chronic Pb-exposed conditions. 85 Antioxidant enzymes maintain balance between pro-oxidant and antioxidant levels. Pb is known to disrupt this balance by binding to sulfhydryl groups at their active site and inactivating it. 86,87 An important antioxidant enzyme is the glutathione-S-transferase (GST) (EC.2.5.1.18), a phase II metabolic isozyme which catalyzes the conjugation of GSH to Pb for detoxification. 88 Several studies have shown an association of Pb-B with intracellular GSH levels irrespective of Pb conjugating either enzymatically or nonenzymatically with GST. The activity of other enzymes in the detoxification pathway is also known to decrease with increasing concentration of Pb in the blood. Studies have shown that variations (deletion/polymorphism) in GST (GSTM1, GSTTI, and GSTP1) produce significant alterations in the activity of GST which may influence interindividual differences in response to chemical and carcinogenic compounds and in the development of multifactorial diseases. 51,52,89 –91 Population studies have demonstrated a link between low-level Pb exposure and subsequent development of hypertension and cardiovascular diseases via oxidative stress. 92 Lee et al. showed a strong association of GST-theta 1 (GSTT1) (Gene ID: 2952; Accession no.: NT_187633.1) positive allele polymorphism with hypertension of Pb-exposed Korean male workers. 93 Further, in vivo and in vitro studies reveal that hypertension and cardiovascular diseases alter the renin-angiotensin system. 94 A study by Jiao et al. showed that occupationally Pb-exposed workers presented higher serum angiotensinogen levels compared to control individuals with low Pb-B levels but found no association of AGT (Gene ID: 183; Accession no.: NC_000001.11) variants, rs699, and rs4762 with Pb exposure. 54 Pb-induced oxidative stress in erythrocytes results in lipid peroxidation followed by membrane fragility and death of the cell. During this process, heme oxygenase-1 is induced both in vitro and in vivo. 95 –97 A previous study showed that HMOX-1L (Gene ID: 3162; Accession no.: NC_000022.11) alleles, HFE H63D, and HFE C282Y variants contribute as susceptible markers of cardiac toxic events from Pb exposure. 98 Another study showed that HFE H63D and GSTP1 Ile105Val polymorphism may have a potential role in modifying effects of Pb on the development of amyotropic lateral sclerosis. 48 Similarly, the CAT (C111T) polymorphism was shown to influence the Pb-B level and blood pressure; hypertensive subjects homozygous for mutant TT genotype was shown to have low catalase activity and higher Pb-B levels compared to the wildtype CC carriers. 55 Variations in oxidative stress related genes such as GPX-1 (Gene ID: 2876; Accession no.: NC_000003.12) (rs1050450, rs1800668), RAC2 (Gene ID: 5880; Accession no.: NC_000022.11) (rs2239774), and XDH (Gene ID: 7498; Accession no.: NC_000002.12) (rs7574920) were found to significantly modify the association of cumulative Pb exposure with risk of glioblastoma and meningioma. 53 Heavy metal exposure alters the methylation pattern of oxidative stress-related genes. Li et al. showed no association between GSTP1 methylation and the risk of Pb poisoning since the recruited subjects in this case were in their early stage of Pb exposure and it was further speculated that it may change during later stages of Pb poisoning. 99
Pb and the immune system
Pb is known to alter health by immuno-modulation. It produces immuno-modulatory effects at concentrations comparable to Pb-B levels of 1–10 µM. 100 Occupational or environmental exposure to Pb results in immuno-toxic effects attributed by immuno-enhancement and or immuno-suppression depending on the magnitude and duration of Pb exposure. Pb is shown to enhance B-cell, T-cell production, and major histocompatibility complex activity in vitro. In vivo and ex vivo animal studies revealed that exposure to Pb could activate responses mediated by T-helper 2 cells and suppress the production of T-helper 1 cells. Lack of T-helper 1 cells diminishes the host resistance. 101,102 Circulating antibody titers against infectious agents were found to be significantly lower in animals exposed to Pb than control animals’ indicative of immuno-compromisation. 103 A marked depression in serum immunoglobulin (IgG, IgM) and complement (C3 and C4) levels were observed in a study of 25 occupationally exposed storage battery plant workers compared to controls. 104 A gender-specific study revealed that males showed a positive association of Pb-B and IgE levels and increased risk of asthma compared to females. 105 However, molecular mechanism of Pb complex and its effect on the immune system remain obscure. Oxidative stress induced by Pb is known to activate transcription factors. Ramesh et al. showed that Pb can activate transcription factors such as nuclear factor kappa B (NFκB), activator protein-1 (AP-1), and kinases like MAPK/ERK kinase (MEK) and c-Jun N-terminal kinases (JNK) in a dose-dependent manner. 106 Taken together, earlier studies have shown that Pb-induced activation of transcription factors may elicit the expression of genes involved in cell proliferation, apoptosis, host-defense mechanisms, and inflammation. Further, Pb has shown to induce the activation of NFκB by binding to its promoter and in regulating IL-6 expression indicating its significant role in immuno-modulation. Pb is further shown to increase T-cell proliferation both in murine and human cells in vitro and in stimulating mixed leukocyte responses both in vitro and in vivo. Lymphocytes challenged with Pb at concentrations of 10 µM or above (equivalent to a Pb-B level of 200 µg/dL) induced IL-8 secretion. Animal studies revealed that Pb targets the T-cells, dendritic cells, macrophages, and neutrophils of which the TCD4+ cells are the most affected. 107 Similarly, Pb-exposed workers with Pb-B levels between 30 and 70 µg/dL showed alteration in T-cell population, T-cell mitogen response, and chemotaxis of polymorphonuclear leukocytes. 108 Natural killer cells on the other hand appeared to be less sensitive to the effects of Pb. 109 Furthermore, Pb can influence the expression of cell surface markers of lymphocyte-specific antigens. Fischbein et al. observed decreased lymphocyte abundance along with decreased CD3+ and CD4+ cells in individuals with slightly elevated Pb-B levels in the range of 15–55 μg/dL. 110 Similarly, Undeger et al. observed a reduction in the number and percentage of CD4+ cells among Pb-exposed workers with a mean Pb-B level of 74.8 μg/dL indicating detrimental effects of Pb on the host immune system. 104 Mishra et al. showed that Pb stimulated interferon-γ production in phytohemagglutinin-mediated peripheral blood mononucleocyte cultures of occupationally exposed workers while the natural killer cells remained unaffected. 111 Pb is also known to induce cytokine production in response to inflammation in occupationally exposed groups. 112 Oxidative stress triggers the signaling cascades of inflammatory processes expressing inflammatory markers such as tumor necrosis factor (TNF-α), interleukins (IL-6, IL-1β), and high sensitivity C-reactive protein (CRP). 56 In a study of Nigerian Pb-exposed workers with elevated Pb-B levels, the immune system was significantly lowered with rise in CRP levels (p < 0.01). 113 Songdej et al. showed that the CRP level in men with Pb-B levels ≥3.09 μg/dL was 77% greater as compared to those with a Pb-B level <1.16 μg/dL. 114 Genetic variation studies have also been performed to understand the influence of immune-related genes on susceptibility to Pb exposure. In a cohort study of nonoccupationally Pb-exposed Thai subjects, it was observed that subjects with GSTT1, GSTM1 (Gene ID: 2944; Accession no.: NC_000001.11) null genotype, and GSTP1 (Gene ID: 2950; Accession no.: NC_000011.10) Ile105Val variant allele was more sensitive to Pb exposure with elevated CRP levels. 77 Two studies have reported the presence of a nucleotide change (G>A) at position -308 of TNF-α (Gene ID: 7124; Accession no.: NC_007130.6) known to modulate TNF-α expression and its influence on individual susceptibility to various illnesses including cancer. 115,116 Kim et al. observed an association of Pb-B level versus elevated TNF-α level among TNF-α GG genotype in nonoccupationally Pb-exposed Korean male subjects with Pb-B levels ≥2.51 μg/dL. 56 Likewise, there is a need to screen for genetic variations in association to Pb-induced oxidative stress and inflammation to understand the impact of Pb on the immune system.
Pb and metal homeostasis
Metals play a vital role in maintaining homeostasis inside the cell by serving as essential cofactors for many enzymes involved in cellular metabolism and growth. 117 Pb represents a major threat for mammalian cells because of its ability to mimic essential metals like Ca, Fe, and Zn in the initial step of transport and metabolism. 118 The permeation of Pb across the plasma membrane into the cell is mediated by channels/transporters which in turn affect the biological function. An in vivo study reported the enhanced expression of metal transporter proteins such as ZIP14 (low/medium doses), CTR1 (high doses), and ZIP8 in liver of Pb-exposed rats. 119 Hence, several modes of interaction resulting in the formation of metal-protein, metal, ligand, and enzyme bridge complexes exist. 120 Here we discuss genes that influence metal homeostasis and its role in Pb toxicity.
Metallothionein
Metallothioneins (MTs) are a group of low molecular weight, cysteine-rich metal binding proteins involved in the maintenance of metal homeostasis in response to heavy metal exposure. They are central to the natural response of the body to toxic metals and are expressed in almost all cells. The metal-binding property of MT makes them very crucial for several biological functions such as the detoxification of accumulated toxic metals, scavenging of free radicals, and in maintaining metal homeostasis. The sulfhydryl (–SH) groups of MT cysteine residues are involved in binding with metals like Zn and Cd. Upon exposure to various heavy metals like Cd, Pb, and Hg, MT codes for the metal regulatory transcription factor-1 (MTF1) which accumulates in the nucleus and bind to the promoter containing metal-responsive elements (MRE) to influence key processes involved in gene regulation, cellular proliferation and differentiation, signal transduction, and apoptosis. 121 Szitanyi et al. showed that at high Pb-B levels, the MT concentration was low indicating that MT alone cannot defend Pb directly unless induced by other factors. 122 A study recently reported that MT1 can serve as a potential marker for exposure to pollutants. 123 Earlier data suggest that only two genes—MT1 and MT2 are expressed in the presence of Pb. MT2A decreases the binding affinity of MTF1 to MREa reducing MT transcription. 124 MT2A polymorphisms have been studied for their association with risk of diseases like diabetes, stroke, and atherosclerosis including prostate and ductal breast cancer. 125 –127 A MT2A polymorphism located in the 5′ region of MREa near the TATA box, -5 A/G (rs28366003) has shown to decrease the induction of gene transcription but influences the increase in MT level in response to heavy metals exposure or cellular stress. Only limited studies are available to assess the influence of MT polymorphism on Pb-B level in occupationally and environmentally exposed subjects. Kayaalti et al. evaluated the influence of MT2A rs28366003 polymorphism (-5 A/G) on Pb-B levels and observed that individuals homozygous for MT2A GG had higher Pb-B levels compared to heterozygous carriers. 37 Further, Tekin et al. investigated the influence of this polymorphism on maternal blood, placental tissue, and cord Pb-B levels and reported that pregnant women heterozygous for MT2A rs28366003 AG variant had higher Pb-B level indicating an elevated risk of low-level cord blood Pb exposure. 39 Another study by Krzeslak et al. reported the association of this polymorphism and the risk of Pb toxicity in prostate cancer and observed that individuals with prostate cancer polymorphic for the GG variant had higher Pb-B level compared to control subjects. 50 Another MT2A polymorphism located in the 3′ UTR region (rs10636) has also been studied in relation to Pb toxicity. Gundacker et al. observed a negative association between MT2A rs10636 C/G variants and Pb-B levels within a group of individuals in Austria. 49 Recently, it was reported that individuals occupationally exposed to Pb who are polymorphic for MT2A rs10636 GG genotype is associated with higher Pb-B level. 128 Several studies like these however report variations in outcome and continues to be ambiguous. MT, therefore, serves as a candidate gene to assess the effect of modification of Pb exposure on MT expression. 129
Solute Carrier Family11 Member 2
SLC11A2 or divalent metal transporter 1 (DMT1), a transmembrane, nonheme protein encoded by SLC11A2 (solute carrier 11 group A member 2) aids in the intestinal absorption of Fe from Fe stores. It is found abundantly in the proximal part of the duodenum and endosomes. SLC11A2 consists of 12 transmembrane domains and an extra cytoplasmic domain consisting of glycosylation signals linked by asparagine. Till date, four mammalian isoforms of SLC11A2 has been identified. Of these, two isoforms are Iron Responsive Elements (IRE) positive and IRE negative resulting from alternative splicing at its 3′ end. The other two isoforms resulting from alternative splicing of 5′ exons designated as 1A and 1B. 130 SLC11A2 transporter mediates the uptake of dietary Fe from the lumen into the enterocytes. The Fe levels in the body are regulated by SLC11A2 since there is no mechanism by which Fe can be excreted out of the system. Following reduction of Fe3+ to Fe2+, it enters the cell via SLC11A2 transporter. From the cell, Fe2+ is transported via ferroportin (Fpn1) into the plasma where it gets oxidized back to Fe3+ and binds to transferrin (TF). The transferrin bound iron (TF-Fe3+) is then transported to the liver through transferrin receptor (TFRC) where Fe is stored and utilized for the synthesis of heme. SLC11A2 therefore appears to be a very crucial transporter for the transport of nonheme iron. Pb and Fe share similar cationic characteristics and hence share similar divalent metal transporter. SLC11A2 is known to mediate the transport of divalent metals like Pb and Cd. 35 Functional characterization studies revealed that SLC11A2 enables increased absorption of Pb and Cd during Fe deficiency. 36 When SLC11A2 was cloned and expressed in Saccharomyces cerevisiae which lacks the machinery for high affinity Fe transport, both Fe and Pb were transported with similar affinities; further it was observed that increased concentrations of Fe suppressed the transport of Pb. 131 A study reported that Pb exposure promoted expression of DMT1 and DMT1-IRE. 119 Further, it was shown that the uptake of Pb by DMT1 was reduced when co-exposed with Fe. 132 Several studies of SLC11A2 polymorphisms have been implicated in Pb toxicity. Till date, five SLC11A2 polymorphisms have been described in the literature which includes 1254 T/C (rs1048230), 1303 C/A (rs144863268), IVS4+44 C/A (rs224589), IVS2+11 A/G, and IVS6+538 G/G of which IVS4+44 C/A, located on intron 4 has been found to be associated with high Pb-B levels. 35 Kayaalti et al. investigated the influence of IVS4+44 rs224589 C/A on Pb-B levels in Turkish population and found that individuals homozygous for CC genotype accumulated more Pb as compared to other genotypes. 133 Another study conducted on Korean population on the same variant by Kim et al. showed that the AA genotype had higher Pb-B levels and heterozygous CA was at increased risk of hypertension. 35 Ngueta hypothesized that non-Hispanic Black individuals with the +1A/IRE+ isoform is most active which is one reason why this section of individuals are associated with elevated Pb-B levels independent of age and socioeconomic conditions. 134
Cross talk between Pb and Ca metabolism
Pb shares similar divalent properties as Ca and its interaction has been identified in several in vitro and in vivo models. Pb enters the cell via one or more Ca channels that is expressed in various cells of the body and hence interferes with Ca-mediated processes by binding to calmodulin, protein kinase C, and synaptotagmin I. 135 –137 Variations in genes associated with Ca regulation may influence the uptake of Pb indirectly. Genes that encode Ca channels such as TRPV5 (Gene ID: 56302; Accession no.: NC_000007.14), TRPV6 (Gene ID: 55503; Accession no.: NC_000007.14) (transient receptor potential cation channel subfamily V), and intracellular Ca chaperones like CALB1 (Gene ID: 793; Accession no.: NC_000008.11) and VDR are shown to regulate the cellular Ca levels.
Vitamin-D receptor
Vitamin-D is involved in various biological processes including bone metabolism, modulation of immune responses, and regulation of cellular proliferation and differentiation. 138 It enhances intestinal and renal reabsorption of Ca by binding to vitamin-D receptor and driving the expression of calbindin-D, a Ca-binding protein to which Pb has higher affinity compared to Ca. 24 Therefore, Pb accumulation in calcifying tissues such as bone and teeth increases. The bone accounts for more than 94% of body Pb burden in adults and 70% in children. 45,139 Studies report that variations in VDR have a genetic control over bone mass and in the toxicokinetics of Pb during bone resorption. 140 To date, several epidemiological studies have reported the influence of five VDR polymorphisms on blood and bone Pb levels including rs11568820 (Cdx-2), rs2228570 (FokI), rs1544410 (BsmI), rs731236 (ApaI), and rs731236 (TaqI). These polymorphisms have been implicated in disease endpoints like osteoporosis and fracture risk, essential hypertension, cardiovascular diseases, cancer, and immune-related disorders. 136 Three adjacent polymorphisms—BsmI, ApaI and TaqI, located on intron 8/intron 9, extending into the 3′ UTR region of VDR is known to affect the mRNA stability and/or protein translation efficiency eventually modulating the receptor binding affinity. Lee et al. showed that Pb workers with BsmI A (GA or AA) allele had higher prevalence of hypertension compared to those with the common GG genotype. 71 Further, Schwartz et al. reported that Pb workers with BsmI A allele had significantly (p < 0.05) higher Pb-B (mean = 4.2 μg/dL), chelatable Pb (mean = 37.3 μg), and tibia Pb (mean = 6.4 μg/g) levels than did workers with the GG genotype. 141 Another study by Wananukul et al. on Thai Pb-exposed workers showed that individuals homozygous for wild GG and mutant CC genotypes of BsmI and TaqI polymorphisms respectively were significantly associated with lower Pb-B levels (p = 0.00; p = 0.01). 29 A study also showed that a combination of ALAD 1-2/VDR BsmI AA play a significant role in Pb-induced nephrotoxicity at low Pb-B levels with continuous exposure. 142 Another study by Pawlas et al. identified that VDR alleles of BsmI B, TaqI t and FokI F polymorphism was associated with impaired hearing in children. 143 The same group also revealed that VDR FokI polymorphism significantly modifies the effect of Pb-B levels on children’s posture. 144 Recently, it has been reported that maternal, placental, and cord Pb-B levels was higher in mothers carrying the f allele of FokI polymorphism. To explain the association of a nonfunctional polymorphism, we rely on linkage disequilibrium. Most of the SNPs are found in linkage disequilibrium in regions called haplotype blocks which is much more informative in association studies. 145 Linkage disequilibrium measures the co-occurrence of alleles of adjacent polymorphisms with each other. 146 To investigate the influence of VDR haplotype on circulating Pb-B levels, a study by Rezende et al. showed that haplotypes combining the common alleles of ApaI, BsmI, and FokI polymorphisms were associated with lower Pb-B, plasma Pb and percentage of Pb-B and plasma Pb ratio indicating that VDR modulates the circulating Pb-B levels. 147
Other Ca binding proteins
Pb mimics essential metals and alters the regulation of cellular metabolism. The effect of Pb on Ca fluxes and Ca regulated events are major mechanisms of Pb neurotoxicity. Studies concerning variations in genes encoding Ca binding proteins (transporters/channels/transcription factors) in Pb toxicity are limited. Two promoter polymorphisms of CALB1, rs1800645 and rs16902897 located within 112 bp of a putative vitamin-D response element (VDRE) at positions -366 and -490 respectively has been reported in a cohort study of male Finnish smokers in association with risk of renal cell carcinoma. 148 In this study, it was observed that higher concentration of whole Pb-B (p = 0.022) and the CALB1-p366 “TT” genotype (p = 0.057) was associated with increased risk of renal cell carcinoma; but the polymorphism showed no influence on Pb-B levels. Two other Ca binding proteins, protein kinase C (PKC) and calmodulin (CaM) is also implicated to play potential roles in Pb toxicity. Pb stimulates CaM and CaM phosphodiesterase and enhances CaM-mediated protein phosphorylation in synaptic vesicles and interferes with the CaM-mediated neurotransmitter release. Several studies revealed that Ca binding sites of Ca binding proteins is highly accessible to Pb ions with higher affinity even at low levels leading to their activation and altering cellular processes involved in transport and metabolism. 149 Nanomolar concentrations of Pb is sufficient to activate PKC which generates a Ca influx increasing the Ca ion stores by stimulating the Ca pore channels. 150,151 However, in the presence of excess Ca, proteins like MGP (matrix-γ-carboxyglutamate) can suppress Ca ion function. Since Pb competes with Ca, Pb can be prevented from deposition by MGP. Shaik et al. studied the influence of promoter MGP polymorphism on Pb-B levels of occupationally exposed workers and observed that the common MGP (rs1800802) TT variant was associated with higher Pb-B levels. 152 Since the function of the protein is altered in the presence of Pb, variations may be induced in genes but with a lower frequency. With available literature on expression of proteins altered by Pb, variations in genes associated with these alterations can be targeted.
Cross talk between Pb and Fe metabolism
Earlier studies have indicated a possible cross talk between Pb and Fe metabolism as the two metals share similar divalent characteristics. Fe is a fundamental nutrient involved in various cellular processes such as in the biosynthesis of heme and in the functioning of ribonucleotide reductase which catalyzes the formation of deoxyribonucleotides. When the synchrony of Fe homeostasis is affected, the normal functioning of a cell is also affected. Interestingly, the physiological Fe status is inversely associated with Pb absorption. 153,154
Hemochromatosis (HFE) protein plays an important role in regulating Fe levels upon its interaction with TFRC and TF. Mutations in HFE have been demonstrated to influence Fe storage leading to a pathological condition called hemochromatosis (also referred to as Fe loading disease), a serious condition that develops when the body absorbs excess Fe over the years and accumulates in organ tissues such as joints, liver, pancreas, heart, and pituitary glands. 24,32,155 Feder et al. identified two missense mutations- H63D (rs1799945) and C282Y (rs1800562) in HFE that results in hemochromatosis. 156 The H63D-mutated HFE protein forms a stable complex with TFRC thereby lowering its binding affinity for TF and leading to relatively less uptake of cellular Fe content while C282Y-mutated HFE protein is expressed with an altered β2-microglobulin hence disrupting its transport and presentation on the cell surface. Individuals with this condition lack functional HFE with increasing TFRC expression and absorption of Fe. Interestingly, individuals harboring mutations in HFE have been observed for susceptibility to increased Pb absorption indicating a strongevidence for a cross talk between Pb and Fe metabolism. 24,30,157 Several association studies revealed the influence of HFE mutations in modifying the impact of cumulative Pb exposure on factors such as cognition, 31 QT prolongation, 98 air particle-related metal transport, 158 blood pressure, 32 atherosclerosis, cardiovascular disease 159 and neurodegenerative end points in elderly men suggesting the influential role of HFE on Pb toxicity in addition to Fe uptake. In a normative ageing study of 2280 male subjects, Wright et al. showed that the H63D and C282Y variants were associated with lower patella and Pb-B levels (p = 0.05). 35 Similarly, another study of 619 subjects revealed that H63D mutation but not C282Y, enhances susceptibility to the deleterious impact of cumulative Pb on pulse pressure. During Fe deficiency, TFRC are expressed on the blood brain barrier to replenish Fe levels providing opportunity for interference of Pb as studies have shown that Pb interferes with TFRC expression and TF endocytosis. 160 –162 Also, studies on transgenic mice with human TF gene revealed that Pb mediated the suppression of TF transgene expression in mouse liver without suppressing the mouse endogenous hepatic TF expression. 163 Further, a study reported the association of TF (rs1049296) TT variant in modifying the effect of Pb exposure and hemoglobin levels on child neurocognition. 38 Another study by Karwowski et al. showed that maternal TF (rs1049296) TT variant affects Pb transfer across the placenta. 164 Further, three TF polymorphisms—rs2715632, rs2715631, and rs2715627 were shown to modify the association of Pb and homocysteine levels suggesting the role of Fe in the effect of Pb exposure on homocysteine levels. Hence, cross talks between Fe and Pb metabolism is evident and cumulative effects of such interaction may result in toxicity.
Pb and its effect on cell cycle and apoptosis
Several studies have shown that sublethal concentrations (5–10 µM) of Pb alters physiological processes that are sensitive in its presence and tend to overwhelm these processes such that incremental effects are seen with higher exposures. This phenomenon is referred to as “hormesis effect” where the cell experiences “low dose stimulation with beneficial effects or high dose inhibition with toxic effects.” 165 Pb has been demonstrated as a weak mitogen. It produces a dose-dependent enhancement of mitogen responses leading to cellular proliferation. 166 Subsequently, studies also show that very low doses of Pb could influence the cell cycle by increasing DNA synthesis. 167,168 Further, it has been shown that low-level Pb exposure induce proliferation of peripheral blood mononuclear cells through the activation of PKC dependent cell signaling cascades. 169 Genes that regulate cell growth and proliferation such as those in the MAPK pathway are directly affected by Pb through the oxidative stress mechanism. Several studies revealed that cellular stimulus including Pb exposure that mediates ROS production could activate MAPK pathways in multiple cell types. 170 –172 Further, sublethal doses of Pb (1–10 µM) have shown to increase phosphorylation of MAPK proteins such as ERK 1/2 and p38MAPK. 173 Interestingly, it was observed that 1 µM of Pb (equivalent to a Pb-B level of 20 µg/dL) activated MEK and JNK in cultured PC-12 cells. 106 Animal studies also revealed that Pb (5–10 µM) activates ERK 1/2 and p38MAPK in in vitro and in vivo conditions. 173 –175 However, such studies revealed a biphasic activation indicating that dose and time of exposure are important factors in triggering a biological function in a cell.
Pb-induced oxidative stress is also known to mediate apoptotic mechanisms mainly through the activation of NFκB and p53. Pb induces apoptosis in organ tissues like brain, testis, fibroblasts, lung, and retinoid cells. 176 Studies suggest that both mitochondria and caspase play a vital role in the initiation and execution of apoptosis. 177 Accumulated Pb can damage the mitochondria and alter the regulation of intracellular Ca concentration. Increased influx of Ca can exacerbate the mitochondrial electron transport by activating hydrolytic enzymes that can increase energy expenditure and decrease energy production leading to production of ROS. It has been shown that during or after exposure to Pb, the expression and activity of rod cGMP phosphodiesterase decreases increasing the permeability of ion-channels, Ca overload, reduction in ATP synthesis resulting in cytochrome c, increased caspase c and finally cell death. 178 In vitro studies reveal that Pb induces mitochondria-dependent apoptosis resulting in increased apoptotic rate, externalization of phosphatidyl serine, Bax/Bcl-2 ratio, up-regulation of caspases 3 and 9 expressions as well as decreased mitochondrial membrane potential. 176,179 Similarly, it has been shown that Pb-induces apoptosis by enhancing the expression of Bax, cleaved caspase-3 protein and decreasing Bcl-2 levels in vivo. 180 –183 In addition to mitochondria-induced apoptosis, emerging evidence have indicated that Pb toxicity is linked to endoplasmic reticulum (ER)-driven apoptosis and autophagy. In the endoplasmic reticulum, Pb is shown to induce Grp78 (Glucose regulated protein/ Binding immunoglobulin protein of 78 kDa), a master ER chaperone in astrocytes and glioma, hepatoma HepG2, renal NRK52 cells, endothelial cells in in vitro 184 –187 and spleen cells in in vivo conditions. 188
Pb and cancer
Pb has been studied extensively for many years but its carcinogenic mechanism is not well understood. The International Agency for Research on Cancer (IARC) has classified inorganic Pb compounds as probable (Group 2B) carcinogens, but organic Pb remains unclassified. 189 Several epidemiological and experimental data confirm that inorganic Pb is associated with increased risk to cancer. 14 Studies on rodent models revealed the formation of Pb induced renal adenomas, lung adenomas and cerebral adenomas when administered with compounds such as Pb acetate, Pb subacetate and Pb phosphate in food and drinking water. Pb induces cellular proliferation in rat kidney and liver. 190 Canaz et al. showed accumulation of Pb in epithelial ovarian cancer and borderline tumor. 191 Epidemiological studies on Pb-exposed workers revealed increased risk of renal, 192 lungs, 193 and brain 194,195 cancers. Fu and Boffetta performed a combined meta-analysis to review the epidemiological evidence of carcinogenicity of occupational exposure to inorganic Pb and they observed a significant risk of stomach cancer, lung cancer, and bladder cancer with relative risk ratios and 95% confidence intervals of 1.11 (1.05–1.17), 1.33 (1.18–1.49), 1.29 (1.10–1.50), and 1.41 (1.16–1.71) respectively. 196 However, Pb induced carcinogenesis continues to be a topic of skepticism due to co-exposures from genotoxic agents such as cigarette smoke or other heavy metals like cadmium (Cd) and arsenic (As) which are often found together with Pb during mining activities (Silbergeld 2003). 197 Studies now show that co-exposure provide evidence for interaction of Pb with other forms of exposure. For instance, Hengstler et al. showed that exposure to Pb alone did not induce DNA single stand breaks but with increase in Pb exposure from 1.6 to 50 µg/m3 in air, in the presence of constant exposure to cobalt and Cd resulted in a 5-fold increase in odds of strand breaks. 198 Similarly, it has been noted that the interaction of Pb with As increases the risk of lung cancer in smelter workers, and co-exposure to Pb and engine exhaust was associated with increased risk of lung cancer. 199,200 A NHANES II cohort study on mortality (a US population—based study to assess Pb exposure and other health risks conducted from 1976 to 1980) also showed a positive interaction between Pb and smoking. Such studies express that Pb is a “facilitative” or “permissive” carcinogen with mitogen activity facilitated through mechanisms of oxidative stress, inhibition of DNA synthesis and repair, interaction with DNA binding proteins and tumor suppressor proteins. 196 This cancer—like activity induced by Pb was confirmed by Lu et al. who showed that inorganic Pb stimulated DNA synthesis. 58 The genotoxic nature of Pb was further evaluated at the genetic level to identify possible candidates responsible for DNA damage in relation to Pb exposure. One study in particular investigated the influence of polymorphisms involved in base excision repair (BER) such as XRCC1, APEX1, MUTYH, PARP1, MPG, PARP4, and OGG1; nonhomologous end-joining (NHEJ) repair such as XRCC4, LIG4, and XRCC5 and homologous recombination (HR) repair such as XRCC2, XRCC3, RAD51, and NBS1 pathways in a group of occupationally exposed Pb workers. 59 Most of these have been previously associated with reduced risk of cancer but has not been evaluated as a predictor of Pb toxicity. The XRCC1 rs1799782 variant (CT/TT) was found to be associated with higher Pb-B levels indicating susceptibility of this polymorphism to Pb toxicity. 201 In the study by Garcia-Leston et al., higher levels of T cell receptor mutation were found to be associated with APEX1 (Gene ID: 328; Accession no.: NC_000014.9) rs1130409 G allele carriers. 59 APEX or apurinic/apyrimidinic endonuclease 1 (EC 4.2.99.18) plays a central role in the BER mechanism and requires Mg ion for performing its exonuclease and endonuclease activity. 202 Pb may substitute for Mg in the protein encoded by the allele and hence become more susceptible to inhibition by Pb. Another protein involved is MUTYH (EC 3.2.2) and it encodes DNA glycosylase. It mainly excises the adenine bases which are inappropriately paired with guanine, cytosine or 8-oxoGuanine. 203 Higher levels of DNA damage were observed in MUTYH (Gene ID: 4595; Accession no.: NC_000001.11) rs3219489 CG variants. 59 Another protein which cleaves 8-oxoGuanine is 8-oxo-guanine glycosylase 1 (EC 3.2.2) encoded by OGG1 (Gene ID: 4968; Accession no.: NC_000003.12). This study reported a significant increase of oxidative damage in homozygous individuals for the OGG1 rs1052133 G variant allele, suggesting a less effective repair of 8-oxoGua DNA lesions. 59 In the final step of NHEJ pathway, two proteins- XRCC4 (X-ray repair cross-complementing 4) and LIG4 (DNA ligase 4) (EC 6.5.1.1) forms a complex involved in the alignment and rejoining of DNA ends. 57 Any change in the amino acid content may result in the diminished stability of complex, low activity and defective DNA repair. The homozygous variant allele carriers of LIG4 (Gene ID: 3981; Accession no.: NC_000013.11) rs1805388 (T/T) and XRCC4 (Gene ID: 7518; Accession no.: NC_000005.10) rs28360135 (C/C) showed higher levels of T cell receptor mutation frequency and DNA damage indicating an association of these polymorphisms and less effective DNA repair activity. 59 Liu et al. reported that heterozygous variant carriers of XRCC3-241 CT/TT had a significantly higher mean Pb-B level than homozygous carriers. 41
Pb as an epigenetic modifier
Epigenetics involves the processes of DNA methylation, histone modification and synthesis of small, noncoding RNA molecules. Homocysteine play an important role in maintaining the methylation processes in the body. Earlier studies have shown an association of plasma Pb and homocysteine levels which is an important amino acid in the methylation process. 204 Heavy metals are known to induce epigenetic alterations and these alterations in gene expression are associated with serious and complex diseases. Several studies have implicated the role of Pb as an epigenetic modifier. Toxico-epigenetics of Pb is a growing area in Pb toxicity research and is gaining pace since its inception in 2009 when the first report on the influence of prenatal Pb exposure on genomic methylation of cord blood DNA was published. 205 From this study came streaming research ideas to further understand the epigenetics and developmental exposure of Pb. However, with advances in “omics technology,” tools to determine if epigenetic changes are occurring in-utero because of maternal Pb exposure can be accomplished. Pilsner et al. reported an association between global DNA methylation in umbilical cord white blood cell DNA and maternal patella Pb levels. 206 Further, global methylation levels of cord blood Alu and LINE-1 markers were analysed in association with maternal patella and tibia Pb levels and it was observed that the patella Pb is associated with decreased LINE-1 methylation while the tibia Pb is associated with decreased methylation in both LINE-1 and Alu methylation. 207 Li et al. observed a significant increase in ALAD CpG methylation level in a group of Pb-exposed battery plant workers compared to unexposed controls. 207 The same research group also revealed a decrease in LINE-1 methylation levels in the Pb-exposed group. 208 Hence, ALAD and LINE-1 can serve as epigenetic biomarkers for the early diagnosis of individuals with pathologic/toxic Pb exposure and it might be used to develop novel preventative approaches. Further, it was observed that elevated Pb-B levels are correlated with global DNA methylation indicating modulation of gene expression by changes in the epigenetic status. 208,209 Similarly, in the ELEMENT (Early Life Exposures in Mexico to Environmental Toxicants) cohort study, an association was observed between DNA methylation of LINE-1 and growth-related genes such as IGF2, H19, and HSD11B2 and biomarkers of Pb exposure—umbilical cord blood, maternal tibia, and patella. 210 Another gene affected by Pb exposure is p16, a cyclin-dependent kinase inhibitor and tumour suppressor gene. It has been observed that individuals exposed to high levels of Pb (51–100 µg/dL) showed complete CpG methylation of CDKN2A/TP16 promoter, but partial methylation in individuals with low Pb-B levels (6–11 µg/dL) and unmethylated in unexposed. 211 Hence, Pb suppresses p16 expression and promotes cell proliferation which contributes to risk of cancer. 212 Studies also report that pregnant mothers exposed to Pb can impart epigenetic modifications on DNA methylation of their grandchildren, a phenomenon known as “transgenerational effect.” These inherited changes may increase disease susceptibility. Such studies raise the idea that early-life Pb exposure may play a role in adult onset of diseases now called the “fetal origin of adult diseases.” 213 Recently, it has been reported that prenatal Pb exposure can result in cardiovascular diseases later in life. Engström et al. reported a promoter methylation of glycoprotein VI which is involved in platelet activation and thrombus formation. 214
Epidemiological studies have provided evidence that below the CDC reference Pb level, early life Pb exposure can cause harmful effects to the developing brain leading to late onset diseases like Alzheimer’s. In relation to this, Bihaqi et al. studied the brain protein expression pattern in early life Pb-exposed nonhuman primates and observed a repression of H3K9ac, H4K8ac, H4K12ac, and H3K4me2 accompanied by decrease in the protein levels of DNMT, MeCP2 and other histone associated proteins. 215 The same group investigated its expression across the lifespan of the rodents and they observed an elevation of MAT2A levels, decrease in histone proteins- H3K9Ac and H3K4me2 (gene activation) and increase in H3K27me3 (gene repression) across the lifespan of Pb-exposed animals. 216 Such studies revealed that early life Pb exposure reprograms the gene expression profiles either by upregulating or downregulating genes through alternate epigenetic pathways contributing to enhancement of neurodegeneration in old age. Another concept which researchers are focusing on is the differentially methylated regions that regulate the monoallelic expression of imprinted genes. 217 Li et al. showed that early childhood Pb exposure is associated with DNA methylation differences in differentially methylated regions of imprinted genes such as PEG3, IGF2/H19, and PLAGL1/HYMAI during adulthood depending on the sex of the individual. 218 In another epigenome wide association study, it was shown that a CpG site in CLEC11A (cg10773601) and DNHD1 (cg24637308) was negatively associated with prenatal Pb exposure and was prominent among female infants than males. 219 It was further speculated that these methylation alterations could be partly mediated by Pb-induced oxidative stress and metabolism alterations. One such protein sensitive to oxidation during the methylation process is TET (Ten-Eleven-Translocase) proteins which convert 5mC to 5hmC. The levels of 5hmC are abundant in the embryonic stem cells and brain and hence crucial in the early phases of development. 220,221 Sen et al. observed that prenatal exposure to Pb can affect the hydroxymethylation profile of high density 5hmC clusters of imprinted genes leading to their altered expression and function in a sex-specific manner and may also serve as potential biomarkers for susceptibility to Pb associated diseases. 222 In addition to alteration in methylation signatures, Pb also altered miRNA expression profiles which in turn may affect the gene networks targeted by miRNA resulting in diverse diseases including cancer. 223 A study conducted by Bollati et al. on peripheral blood leukocytes showed that miR-222 expression was positively associated with Pb exposure while miR-146a expression was negatively associated. 224 Since Pb is a potent neurodevelopmental toxin, miRNA’s associated with neurophysiological and neurodegenerative pathways were studied. Masoud et al. observed that exposure to Pb acetate in early life produced a transient increase in the expression of Alzheimer disease related miR-106b, miR-29b, and miR-132. 225 Chronic exposure to Pb also resulted in the upregulation of miR-34b, miR-34c, miR-204, miR-211, miR-448, and miR-449a, and the drastic downregulation of miR-494 in the brain. 226 Plasma miRNA profiling on occupationally exposed Chinese workers revealed that miR-520c-3p, miR-211, and miR-148a were down expressed and miR-572 were highly expressed in workers with high Pb exposure and high Pb-B levels. 227 Hence, variations in miRNA expression associated with Pb exposure can serve as determinants for individual sensitivity to Pb poisoning. Table 2 illustrates the methylation signatures and gene expression pattern in response to Pb exposure.
Gene expression and methylation profiles in Pb exposure studies.a
Pb: lead; NAS: normative aging study.
aThe em dash sign indicates no information available. The methylation and gene expression status mentioned here is with respect to high Pb levels only.
bA US-based prebirth cohort.
cEarly life exposures in Mexico to environmental toxicants.
Biomarkers of Pb exposure, susceptibility, and effect
Biomarkers constitute a signal on the level of contamination and toxicological risk. 228 The extent of Pb contamination depend on factors such as valency, solubility, dosage, duration of exposure, and other endogenous factors like age, gender, diet, lifestyle, ethnicity, socioeconomic status, environmental and occupational exposure. 229 Valency of a metal influences the process of biotransformation involving absorption, distribution, metabolism and elimination (ADME) of the toxic substance from the body. 16 The National Research Council has classified biological markers into three types- markers of exposure, effect and susceptibility. 230,231 Biomarkers of Pb will be helpful in evaluating exposure in contaminated regions and implementing health risk assessment. 232
Biomarker of exposure is defined as the measurement of a toxic substance or the product of interaction of a toxic substance and a target molecule of the cell in a compartment within an organism. 228 Whole blood, plasma, urine, bone, and hair constitute biomarkers of exposure. The half-life estimate of Pb in blood and soft tissues is approximately 4 weeks while in the bone it is 20–30 years. About 99% of Pb binds to erythrocytes while the remaining 1% resides in the plasma which constitutes the metabolically active center of the body Pb pool. 233,234 About 80% bound to erythrocytes binds to δ-ALAD, an enzyme sensitive to Pb inhibition. 86,235 Pb-B is considered widely as a classic biomarker for assessment of Pb toxicity and risk. 112 The relationship of Pb-B and Pb exposure is curvilinear. In recent years, various attempts have been made to estimate Pb levels in plasma (Pb-P) but was found to be less sensitive due to issues such as short half-life of Pb-P in less than an hour or contamination by hemolysis during sample collection. 236 Further, it was reported that <5% of total Pb in blood is bound to plasma proteins and only 0.01% of plasma is bioavailable. Analyzing bone Pb levels might also provide a more accurate picture than other biomarkers as it reflects the cumulative body Pb burden of an individual. 140 Some amounts of absorbed Pb are excreted in the urine and this depends not only on the exposure conditions but also on the extent of body burden and kidney function. Biomarkers of effect constitute the measurable biochemical, physiological, behavioral or other alterations within an organism. Biomarkers of Pb and its associated effects depends on the organ system affected. The hematopoietic system affected by Pb is associated with low ALAD activity, increased accumulation of aminolevulinic acid (ALA) in blood (ALA-B), plasma (ALA-P) and urine (ALA-U), urinary coproporphyrin (U-CPP) and ZPP in blood, 237 increased accumulation of pyrimidine nucleotides within red blood cells as a result of inhibition of pyrimidine-5’-nucleotidase activity; 238 –241 increased accumulation of nicotinamide adenine dinucleotide in red blood cells due to impaired activity of nicotinamide adenine dinucleotide synthetase. 242 Pb-induced oxidative stress biomarkers in blood and tissues such as brain and liver include reduced glutathione, high malondialdehyde and decreased activity of glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. Metabolic enzymes such as aldehyde dehydrogenase, tryptophan 2, 3-dioxygenase, malic enzyme, aspartate β-hydroxylase, prostaglandin reductase, two members of aldoketo reductases—AKR1CI and AKRC2 and metal-binding proteins such as MTs are also documented to be up-regulated by Pb; hence, serves as biomarkers of effect. Other activated genes include the transcription cofactor pirin, an Fe binding nuclear protein involved in apoptosis; two members of the amino acid/polyamine transport system, SLC12A8 and SLC7A11/DMT; and KIT ligand, the ligand of the tyrosine-kinase receptor, implicated in pigmentation and cancer and transmembrane proteins. Pb ions form complexes with bioligands like amino acids, peptides or proteins and result in dramatic physiological consequences. A study revealed that workers occupationally exposed to Pb showed higher amino acid levels compared to nonexposed workers. 243 A knowledge of which amino acids are elevated during high Pb exposure will enable us to understand the type of linkages involved in Pb-protein interaction. The crystallographic structure of four Pb-amino acid complexes (Pb-valine, Pb-isoleucine, Pb-phenylalanine and Pb-arginine) has been characterized in the solid state, solution state and gaseous phase. 244 Amino acids associated with the cell membrane might also serve as Pb related biomarkers. Pb binds strongly and effectively irreversible to phosphatidylserine displacing Ca and Mg ions thus blocking cognitive processes- a prominent feature observed in children with Attention Deficit Hyperactivity Disorder (ADHD). Similarly, Pb disrupts the normal sleep-wake cycle by disturbing the serotonin synthesis and altering the activity of several enzymes involved in tryptophan metabolism. Such studies provide scope for the identification of amino acids in Pb associated pathologies. Recent studies have shown that host-gut microbiome metabolic interactions are disturbed by heavy metals including As, Cd, and Pb. 245,246 Multi-omic approaches of the gut microbiome of Pb-exposed mice revealed that numerous metabolic pathways including vitamin E, bile acids, nitrogen metabolism, energy metabolism, oxidative stress, and the defense/detoxification mechanism were significantly disturbed by Pb exposure. 247 Biomarkers of susceptibility are indicators of an inherent ability of an organism in response to exposure. The susceptibility of an individual to Pb poisoning is contributed by genetic factors and all possible genes has been covered in previous sections. However, several studies need to be performed to identify vulnerable genes associated with Pb exposure and disease. Figure 2 represents a block diagram of the role of biomarkers in risk assessment of Pb poisoning. Hence, a comprehensive knowledge of biomarkers of effect, exposure and susceptibility of Pb will aid in the initiation of risk assessment and proper decision making in management of Pb poisoning.
Schematic representation of biomarkers of exposure, effect, and susceptibility for the assessment of Pb toxicity. Pb: lead.
Influence of genetic and epigenetic markers of Pb in risk assessment
In today’s world, with the increasing number of contaminated sites and the discovery of new diseases, risk assessment has become very essential to control the factors associated with causing these risks. The process of risk assessment involves the identification of hazard followed by analysis and evaluation of the hazard and finally ways to eliminate or control the hazard. Risk assessment of Pb for quality of soil, water, food, and population health in selected areas has been performed primarily, but risk assessment based on markers of Pb has never been executed. Several markers of Pb have been identified to date – genomic, epigenomic, and metabolomic. Regulatory bodies such as the Food and Drug Administration (FDA) and Environmental Protection Agency (EPA) are recognizing the potential of this big data and encouraging its submission to establish guidelines and protocols for incorporation in regulatory decision making. 248 However, the integration of big data across chemical and biological space to develop mechanistic pathways and networks remain limited. Hence, it remains a challenging task for traditional toxicologists, risk assessors and risk managers to decipher meaningful and useful biological information from the generated data. Currently, several public and commercial databases are available to provide effective and flexible information of toxico-genomic data complemented with ADME, histopathology, clinical chemistry, and toxicity data. 249 But it lacks information of disease markers in association with Pb toxicity. Such data can help fill the gaps in Pb exposure studies to further understand Pb interaction pathways. Several nongovernmental organizations have taken up initiatives to clean Pb contaminated sites in different parts of the world and has successfully completed the projects. Toxicologists need to look at the big picture that the data holds by collaborating with such institutions and helping in implementing big data and suggesting meaningful strategies for quantitative risk assessment in those regions. Hence, the significant challenge is (1) to comprehensively integrate the disparate chemical, biological, toxico-genomic, -epigenomic and -metabolomic data to elucidate the mechanism of action of Pb and develop quantitative models that are capable of accurately predicting threshold which can further aid in the management of health and disease and (2) collaboration of toxicologists and stakeholders to implement strategies for risk assessment based on the big data. By following such practices, the confidence and consistency of risk assessment increases which may lead to individualized or personalized interventions.
Conclusion
Pb is a contaminant of global concern and continues to be a cause for adverse health problem of significant importance. The risk assessment of Pb poisoning and subsequent decision making is often hampered due to wide variations in exposure and health effects. This draws our attention to ecogenetics which comprises of toxico-genetic and -epigenetic approaches to identify environmental and genetic determinants of Pb exposure and hazard. The toxico-genetic and -epigenetic data from genome wide SNP mapping and toxico-genomic data from global gene expression profiling can collectively facilitate the identification of genes and pathways to determine susceptibility to Pb. Variations in markers associated with mechanism of action of Pb that affect either activation or detoxification are potential candidates to identify susceptible subgroups of populations. Rapid technological advances in the laboratory has also enabled us to develop a deeper understanding of molecular and functional genetics in toxicology followed by epigenetic processes which may serve as an interface between individual’s environment and the fixed genome. This approach may help in parallel to identify and validate biomarkers (of exposure, susceptibility, and effect) that can be used in health and disease management. The role of Pb and its association with other diseases is limited; hence, high throughput (HTP) technology can be applied to identify novel genes, epigenetic mediators, and transcription factors, adding to the existing knowledge of toxic effects of Pb. HTP is highly expensive and cannot be routinely practiced in a clinical setting, but the big data that it generates will test new hypothesis for future research and will aid clinicians in the risk assessment and decision-making process. It is further advisable that interindividual variations in response to Pb toxicity/ exposure be evaluated in different populations region wise to avoid biased outcomes. Further, Pb-associated genetic and epigenetic data generated can be incorporated into the risk assessment process to help genetically sensitive individuals avoid exposure to Pb which may lead to individualized or personalized interventions. This data may further enable clinicians and the scientific community to refine uncertainty factors and address risk assessment of Pb through legal policies.
Footnotes
Acknowledgements
The authors acknowledge TIFAC-CORE, Government of India and Manipal Academy of Higher Education for the infrastructure. We thank Prof. K Satyamoorthy, Director, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, India, for the valuable support and encouragement. Ms. Monica Shirley Mani was provided with fellowship from Dr. T.M.A Pai foundation, Manipal Academy of Higher Education (131700103) and Directorate of Minorities (Award no. DOM/FELLOWSHIP/CR-84/2017-18).
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
