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The increasing exposure of human populations to excessive levels of metals continues to represent a matter of public health concern. Several biomarkers have been studied and proposed for the detection of adverse health effects induced by lead (Pb), arsenic (As) and manganese (Mn); however, these studies have relied on exposures to each single metal, which fails to replicate real-life exposure scenarios. These 3 metals are commonly detected in different environmental, occupational and food contexts and they share common neurotoxic effects, which are progressive and once clinically apparent may be irreversible. Thus, chronic exposure to low levels of a mixture of these metals represents an additive risk of toxicity. Building upon their shared mechanisms of toxicity, such as oxidative stress, interference with neurotransmitters and effects on hematopoietic system, we address putative biomarkers, which may be assist in assessing onset of neurological diseases associated with exposure to this metal mixture.
Pollution is a worldwide problem with immeasurable health consequences, resulting in overexposure to toxic metals. Globally, environmental contamination of heavy metals continues to grow and exposure in the general population, by air, food or drinking water has become an increasing global phenomenon [1–3].
Metals are often introduced into the environment as mixtures . Accordingly, the U.S. Environmental Protection Agency (EPA) recommends greater emphasis on understanding the combined toxic effects of metals [4, 5]. To date, emphasis has been focused largely on single metal exposures [6, 7]. Given that mixtures are commonly viewed as a simple collection of chemicals, understanding the magnitude of toxicological interactions has yet to be fully appreciated . Notably, the practical consequence of studying single metal exposures likely underestimates their health risks in affected populations [6, 8].
Exposures to metal mixtures may synergize or be additive thus leading to adverse health effects exceeding those noted upon exposures to a single metal [9, 10]. Furthermore, while low doses of a single metal may not cause health effects, when combined with other metals, they may present increased health risk . This matter was recently recognized by the U.S. EPA as a key gap in metal risk assessment .
Given the almost infinite number of chemical mixtures, regulators are faced with the problem of how to assess and regulate their toxicity [6, 12]. Recently it has been argued that grouping criteria should focus on common adverse outcomes, with less emphasis on similarity of mechanisms , and that attention should be focused on those having the greatest potential impact on human health . In addition, for practical purposes, criteria are needed to define the relevant components of a mixture. Such criteria cannot rely simply on the concentrations of the compounds in the mixture, but must also take note of their expected contribution to relevant endpoints of toxicity . In this context exposure to neurotoxic agents represents a concern of high priority, given the increased prevalence of a number of neurodegenerative diseases [13, 14].
A recent assessment on the global health impacts of contaminants, identified As and Pb among the six most toxic pollutants threatening human health . Mn is also of great environmental and public health significance due to its broad usage in the ferroalloy industry .
Pb is naturally present in the earth’s crust . Its anthropogenic releases to ambient air from gas, coal, oil and waste have persisted, representing a continued health risk. It is estimated that body burdens of Pb in humans in the modern era are 1000-fold higher than during the preindustrial era . Fortunately, over the last few decades, Pb emissions in developed countries have markedly decreased, largely due to the abolishment of leaded gasoline, which has been paralleled by a decrease in blood Pb levels in the general population, especially children . However, since Pb is a persistent element  and given its long half-life, past emissions remain a matter of concern. Airborne Pb can be deposited in soil and water, reaching humans via the food chain. Drinking water is also a potential source of Pb exposure . Occupational cohorts may be exposed to Pb in the process of manufacturing of batteries, sheet lead, bronze plumbing, ceramic glazes, caulking, radiation shields, circuit boards and military equipment , as well as the glass industry .
The metalloid As is particularly difficult to characterize as a single element because it has a complex chemistry and there are numerous As compounds, with different oxidation states, and differential toxicities. This element naturally occurs in rocks, soil, water and air [19, 21] where it is largely present in the trivalent or pentavalent forms; the trivalent form is the most toxic. Organic As compounds may also be trivalent or pentavalent and they occur in methylated forms; when compared with inorganic As, the methylated metabolites are less reactive with tissue constituents, less toxic upon acute exposures, likely reflecting their fast excretion in urine [22, 23]. Arsenical compounds are known since primordial times as poisons [21, 24, 25]. Exposure to As-contaminated water is a major concern in several Asian countries, where groundwater may be highly contaminated with As [19, 26, 27]. Smelting of non-ferrous metals and the production of energy from fossil fuel represent two major industrial processes that lead to As contamination of air, water and soil. Occupational exposures to As occur as well in several industries, particularly in nonferrous smelting, electronics, wood preservatives, glass manufacturing and application of arsenical pesticides .
Mn is the 12th most abundant element in the earth’s crust  and is an essential element. Few cases of Mn deficiency have been reported in humans, its deficiency can lead to serious health disorders . Excess Mn may cause a wide range of deleterious effects . Diet, contaminated drinking water and inhalation represent the main sources of exposure . In recent years, Mn has received increased attention, since organic Mn has been used as an antiknock gasoline additive - methylcyclopentadienyl manganese tricarbonyl (MMT) in several countries [30, 31]. The most prominent occupations where Mn exposures are common include mining, ore-crushing, ferromanganese production, smelting and welding . Mn is also found at high concentrations in various other industrial settings, such as the manufacturing of dry-cell batteries, production of Mn-containing organic pesticides, fireworks, ceramics, glass, leather, textiles, paint and cosmetics [2, 33].
Despite the existence of a reasonable amount of information pertaining to the health effects induced by Pb, As or Mn exposure [17, 20, 26], the majotiry of the studies focus on solitary exposures to these metals. However, Pb, As and Mn are frequently found as mixtures [34–38], and whenever mixtures are treated as a single complex substance, uncertainties may range from inexact descriptions of exposure to inadequate toxicity outcome information . Therefore, the toxic effects induced Pb, As and Mn alone and also as binary mixtures, will be described.
Pb is a potent health hazard  that may cause neurotoxicity, hypertension, anemia, renal impairment and interfere with sperm production [16, 20, 40]. Pb compounds may also be carcinogens . The metalloid As can induce cancer, genotoxicity and affect the hematopoietic system, liver, kidneys, skin and brain . Exposure to As has also been reported to increase the risk of cancer in skin, liver, bladder and lungs. Long-term Mn exposure is commonly associated with central and peripheral nervous system disorders. Reproductive outcomes might be affected, with decreased libido, impotence and sexual dysfunction. There is no evidence that Mn causes cancer in humans or animals .
Exposure to high Pb levels may result in acute encephalopathy with compromised blood-brain barrier (BBB) function . This structure is highly vulnerable to the toxic effect of Pb and once damaged, Pb itself and other toxicants can easily enter in the brain . In vivo experiments have established that the cerebral cortex and the basal ganglia are affected by Pb [44, 45]. Because Pb exposure may affect several neurotransmitters (dopaminergic, cholinergic and glutamatergic), most frequently early symptoms of human Pb neurotoxicity include irritability, fatigue, depression, headache, decreased attention, memory loss and low-level cognitive impairment . Other concern related to chronic Pb exposures is associated with the propensity of Pb to accumulate in bones  where it has a long half-life. Accordingly, bone acts as a reservoir for Pb with continuous release of this metal, when bones are remodeled .
To date, limited information exists on the effects of As exposure on cellular and molecular mechanisms of the induced neurological effects . This metal easily crosses the BBB. The basal ganglia are particularly vulnerable; As can also have marked effects on the hippocampus and cortex . As exposure has been suggested as a risk factor for Alzheimer’s disease (AD)  as well as Parkinson’s disease (PD) . Children exposed to As show impaired learning and memory, sleep disturbances, abnormal performances and altered latency of auditory evoked potentials [48, 50]. Patients with occupational exposure to As may develop encephalopathy, characterized by impairments of higher neurological functions, such as learning, memory and concentration [21, 50].
Epidemiological data suggest that high Mn concentrations in drinking water may be associated with neurological impairment . Nevertheless neurotoxic effects induced by Mn in humans emerge mostly subsequent to inhalation exposure  where the metal can enter the brain through the olfactory pathways, providing a direct path into brain tissue [32, 52]. Inhaled Mn at exceedingly high levels may lead manganism, which is characterized by motor and postural signs consistent with those inherent to Parkinson’s disease . The subclinical deficits observed in Mn-exposed subjects are consistent with damage of the basal ganglia . In the early stages of intoxication the symptoms may be reversible .
Practically no reports are found on the neurotoxic effects induced by the mixture of Pb, As and Mn, except a few experimental studies on binary mixtures, demonstrating interactions between these metals. The co-administration of Pb and Mn to rats has been associated with reduced brain weight to a greater extent than upon exposure to either metal alone . Pb levels were also increased in several brain regions, such as the cerebellum, cerebral cortex, corpus striatum, hippocampus and midbrain, as compared with the administration of Pb alone . ln addition, Pb increased the concentration of Mn in corpus striatum and midbrain as compared with Mn alone [12, 56]. Low doses of Pb plus Mn raised the striatal Mn and Pb concentrations . Behavioral changes were also observed, such as a significant hypoactivity and aggravation of Pb-induced aggressive behavior . Tests on learning ability, where conditioned avoidance responses were measured, showed that a mixture of Pb and Mn impaired learning to a greater extent than the single administration of Pb. These results suggest a synergistic effect of binary metal mixtures even at subclinical levels . Accordingly, the EPA highlighted the need for further refined assessment of the potential hazard to public health of combined Mn/Pb mixtures, with emphasis on neurological endpoints . In humans, a positive correlation between Mn and Pb in blood was detected, while co-exposure to environmental Pb and Mn has been shown to affect the intelligence of school-aged children . Several studies were performed with other binary mixtures, such as Pb and As, resulting in increased Pb brain levels, along with decreased As concentrations . Neurotransmitter changes were also noted, which were absent when animals were treated with either metal alone . The mixture of As and Pb was shown to cause neuropsychological effects in children living in Morales (Mexico); however, no conclusive results were reached regarding the interactions between these metals . Several studies have also addressed the effects of a mixture of As and Mn , noting that As and Mn exposures led to a greater accumulation of these metals in rats’ brains when compared to animals exposed to single metals. In addition, higher hair As and Mn levels in humans were associated with significantly lower scores on IQ test, verbal learning and memory. In several cases, a significant interaction was noted for Mn and As . With respect to the mixture of the three elements one study reported that when combined, Pb, As and Mn caused changes in neurotransmitter levels. The authors concluded that the findings were complex, but the data supported the concept that co-exposure to multiple metals may cause neurotoxic effects which is absent with exposure to a single metal at similar doses .
Oxidative stress is a shared mechanism of Pb, As and Mn toxicity [61–64, 40]. The 3 metals can generate reactive oxygen species (ROS) [23, 61, 64–67] and affect ROS-metabolizing enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) [20, 23, 40, 61, 66, 68–70] and interfere with cellular redox status, characterized by reduction in intracellular glutathione (GSH) levels [61, 66, 23, 69]. Decreased levels of reduced GSH levels render cells more susceptible to oxidative damage  and may sensitize cells to toxicity associated with other metals .
Several chemicals inhibit acetylcholine esterase (AChE), causing neurotoxicity  via ACh accumulation in the synaptic cleft with an ensuing overstimulation of muscarinic and nicotinic ACh receptors. AChE is anchored to the plasma membrane and there is evidence that lipid peroxidation arising from oxidative stress may alter its activity [72–74]. Indeed, and although much less studied than the dopaminergic system, cholinergic functions are altered by Pb , As  and Mn . Block of ACh release by Mn and Pb has been proposed as a putative toxic mechanism for interference with cholinergic functions [18, 75]. Furthermore, Pb, As and Mn can affect dopaminergic functions [37, 40, 76–80]. Several studies have demonstrated that interference with neurotransmission in cholinergic or dopaminergic neurons is accompanied by oxidative events [40, 48, 65, 74, 77, 81, 82].
Pb, As and Mn exert several effects on the hematopoietic system [83–85], including interference with heme synthesis [40, 62, 86]. Several enzymes that catalyze the heme synthesis metabolic pathway [87, 88] are susceptible to As and Pb [89–91], secondary to their affinity for the functional thiol (-SH; sulfhydryl) groups [61, 92]. Limited information exists on its potential to alter the activity of heme synthesizing enzymes , but it has been established that Mn can alter heme metabolism in an in vitro assay in rat neurons . Interference with heme biosynthetic pathway is commonly characterized by excessive accumulation and excretion of delta-aminolevulinic acid (ALA) and/or porphyrins [95, 96]. Because individual porphyrins differ in their side-chain substituents, different metals may induce signature-specific changes in porphyrin excretion patterns .
Delta-aminolevulinic acid dehydratase (ALAD) is highly sensitive to Pb and As by virtue of its sulfhydryl moiety, and the inhibition of ALAD leads to the accumulation of delta-ALA [61, 63]. At physiological pH, metal catalyzed autoxidation of delta-ALA generates a carbon centered radical (ALA•) as well as O2−•. The ALA• further oxidizes to the imino form of delta-ALA, producing O2−•, which dismutates to H2O2 [40, 63, 86, 97, 98]. Delta-ALA-generated O2−• also promotes Fe release from the endoplasmic reticulum, which may contribute to the generation of •OH through the Haber-Weiss reaction . Furthermore, the coupled autoxidation of delta-ALA with oxyhemoglobin [Hb-Fe (II)] produces methemoglobin and H2O2 . Disruption of mitochondrial membrane potential and promotion of Ca2+ (II) release from the intramitochondrial matrix, with subsequent mitochondrial damage may also occur [95, 99].
Pb can also interfere with coproporphyrinogen oxidase and ferrochelatase (FECH) [97, 63, 100,] and coproporphyrinogen oxidase is also affected by As . Porhyrins, when in excess may also act themselves as toxins [87, 101] given their lipophilic nature and propensity to generate ROS upon exposure to light (430–635 nm), via photochemical reactions [98, 101]. Porphyrins may also form a porphyrin anion radical, which may substitute for O2−• in Fenton-type reactions or produce O2−• by reducing O2 . These events may cause oxidative damage to membrane components, with proteins and lipids as the main targets of photo-damage, leading eventually to cell death [101, 102].
Neurological diseases induced by the exposure to neurotoxic agents are most frequently chronic progressive disorders, where traditional early warning symptoms of the development of the disease may be lacking. They may be only clinically apparent long after exposure to the initiating factor/s, at a point where the disease is irreversible . Accordingly, great effort has been made to identify neuropathological and biochemical biomarkers of neurotoxicity as early indicators of disease [27,103]. A common concept underlying biomarkers of effect is that they should reflect early and reversible biochemical modifications that precede structural or functional damage and may also be predictive of later responses. Still, challenges and imitations exist concerning biomarkers of neurotoxicity .
To date several biomarkers have been studied and proposed for exposure and susceptibility assessment, as well as the detection of adverse health outcomes induced by Pb, As or Mn
Biomarkers of exposure are often obtained from measurement of the parent compound or its metabolites in biological samples . Determination of metal levels in biological samples has been the most common surrogate of exposure  with body burdens generally determined in blood or urine .
Biomarkers of exposure to Pb are characterized by measurements of total Pb levels in tissues or body fluids, and from these, blood Pb concentration is the most widely used parameter in general clinical practice and public health surveillance [16, 107, 108]. The elimination half-life of Pb in blood is approximately 30 days, and therefore, Pb concentration in blood reflects mainly the exposure history of the previous few months .
Several attempts were made to use urinary Pb as a surrogate of blood Pb levels [16, 108], but caution is advised: i) urinary Pb concentration exhibits relatively high intra-individual variability even at similar concentrations of Pb in the blood [16, 109]; ii) estimation of Pb in blood from urinary Pb may be possible on a group basis, but has limited value on an individual basis ; iii) for low level exposures urinary Pb levels are close to the detection limit of the analytical methods; iv) Pb urinary excretion reflects mainly recent exposure; and v) the determination of Pb in urine is further complicated by altered kidney function, in association with the nephrotoxic Pb effects .
The metalloid As is rapidly cleared from blood, which is the reason why measurements of blood As reflect recent exposures or exposures to high As levels [26, 110]. Urinary As measurements have been considered more reliable than blood, because urinary elimination is the major route for the excretion of this metalloid [26, 110, 111]. Therefore, urinary As has been the most used biomarker in epidemiological and occupational studies as indicator of recent As exposure . Speciation of urinary As may also indicate the extent of past cumulative exposure to the metalloid, more specifically by measuring the levels of monomethylarsenic (MMA) and dimethylarsenic (DMA) , resulting from metabolic methylation of ingested or inhaled inorganic As . Total urinary levels of As are used as exposure biomarkers .
Mn levels in biological samples, such as blood and urine, have been investigated as biomarker of exposure , but their suitability is highly controversial . While they may indicate average levels of exposure on a group basis  they are not suitable for individual assessment [17, 113, 114]. Another limiting factor is the rapid rate of Mn clearance from the body, because excess Mn in blood is rapidly removed by the liver and excreted into the bile. Thus, levels of Mn in blood have been used as an indicator of recent exposure, generally less than one month [17, 28, 107]. Since the urinary excretion of Mn is approximately 3% of total excretion urinary Mn has limited clinical validity [107, 28]. Some authors recommend that urinary Mn should be abandoned as a biomarker of exposure .
Biomarkers of susceptibility reflect an inherent or acquired ability of an organism to respond to the challenge of exposure to a specific chemical substance .
It has been known that there is large variation in susceptibility to Pb exposure, with some individuals experiencing toxic effects at levels that others can sustain without any ill effects. Some of these differences may have a genetic basis, namely polymorphisms in ALAD, which is the major Pb-binding protein in the blood [116, 117]. Indeed ALAD2 carriers have a lower risk of toxic effects than ALAD1 homozygotes at the same level of exposure, and some authors suggest that the effects of Pb on neurobehavioral functions tends also to be worse among ALAD1 homozygotes .
Much of the susceptibility to As-related health effects is determined by large interindividual variation in As methylation . The trivalent metabolites (particularly MMA III) are most commonly associated with adverse health outcomes and the distribution of urinary As metabolites is determined to a greater extend by genetic variations and is illustrated by the aggregation of methylation patterns within families [120, 121]. Mn secretion in the bile may vary according to individuals’ genotypic status. A significant association between the CYP2D6*2 polymorphism (a biomarker of susceptibility) and the latency of chronic Mn poisoning has been reported .
The knowledge on the toxicity mechanisms of metals at tissue, cellular and molecular levels are crucial for the discovery of new biomarkers. On the basis of oxidative stress, dopaminergic, cholinergic and heme synthesis alterations induced by Pb, As or Mn, several potential biomarkers of effect are summarized in Table 1 and will be described below.
Serum prolactin (PRL) has been mentioned as an indicator of the dopaminergic function, which is a target of Pb . While Pb’s ability to affect PRL levels has been inconsistent across various studies, PRL is generally increased by exposure to this metal [80, 124]. Pb exposure has been also positively associated with PRL levels in occupationally exposed men . However, in other studies PRL levels failed to change upon exposure to Pb .
The effects of Pb on cholinergic functions have been consistently reported, with alterations preceding the well-known neurobehavioral and neurophysiological endpoints associated with Pb neurotoxicity . It was suggested that AChE activity in erythrocytes may serve as a peripheral surrogate dose–effect index of neurotoxicity on cholinergic function in occupational exposure to Pb ; indeed, decreased AChE erythrocytic levels were observed in workers exposed to this metal . Free radicals can decrease AChE activity, leading to speculation that the inhibitory effect of Pb on AChE observed in the workers may be due to free radicals produced by Pb . Pb effects on heme synthesis are well documented [125, 126] and delta-ALA and ALAD levels in blood are considered the most reliable indicators of Pb intoxication [40, 127, 128], even at relatively low Pb blood concentrations . Since inhibition of ALAD activity results in elevated delta-ALA levels in blood, consequential increase of urinary delta-ALA excretion is expected, and in fact, urinary delta-ALA has been used in clinical diagnosis of chronic occupational Pb intoxication . However, urinary delta-ALA does not seem to be a sensitive indicator when Pb exposure is low . Patients with Pb poisoning also show accumulation of porphyrins due to the inhibition by Pb of heme biosynthetic enzymes. These enzymes are ALAD, coproporphyrinogen oxidase and FECH [63, 97, 100]. Typically increased urinary excretion of coproporphyrin, as well as accumulation of protoporphyrin in erythrocytes is observed .
There are practically no studies evaluating the effects of As exposure on PRL release, but a few have documented that exposure to As leads to reductions in dopamine (DA) content in brain areas involved in the regulation of PRL release  and recent reports mention that sodium arsenite can cause a reduction in plasma PRL levels . Both arsenite and arsenate can cause cholinergic dysfunctions in rats in a dose dependent manner, with concomitant AChE inhibition [21, 133, 134] and consequent disturbances in nervous activity . Increased blood ALAS levels  and changes in ALAD activity have been noted upon exposure to both As (III) and (V)  and it has been posited that ALAD activity in blood may be used to estimate its enzymatic brain activity . Increases in urinary coproporphyrins were found in smelter workers exposed to arsenic trioxide dust, but the mean concentration of uroporphyrin was analogous to that of controls . In contrast, increased urinary uroporphyrin and coproporphyrin levels were observed in individuals exposed to As released from a burning contaminated coal . Further studies are required to clarify this apparent discrepancy in the effect of As among human populations .
The investigation of Mn effects on PRL has led to inconsistent results. Serum PRL has been proposed as a biomarker of occupational Mn exposure , reflecting Mn’s effect on DA neurotransmission . Negative results have also been reported [17, 55, 139]. Some salient features of manganism, such as the intensity of mood disturbances cannot be explained only by the disruption of dopaminergic systems, and likely encompasses the cholinergic system as well. The latter is known to play a crucial role in modulating emotional response and higher cognitive functions. . Miners chronically exposed to exceedingly high levels of Mn may exhibit changes in the activity of blood AChE and ACh , as well as red blood cells (RBC)-AChE . Inconsistent finding exist regarding Mn-induced hematological effects. In vivo studies in rats exposed to Mn by dietary intake have documented decreased hematocrit and hemoglobin levels . Even less information is available on Mn’s propensity to affect heme biosynthetic pathway. One such study suggested that Mn can interfere with heme biosynthesis by inhibiting ALAS activity . In addition, Mn has been shown to inhibit liver and erythrocytes ALAD  and competitively inhibit FECH .
In real life scenarios people are co-exposed to an almost infinite number of chemical mixtures and thus, it is barely impossible to determine all the components of a mixture present in an environmental setting . For practical purposes, if mixtures’ components are not usually well known, criteria are needed to define the relevant components of a mixture. Such criteria cannot rely simply on the concentrations of the compounds in the mixture, but must also take note of the expected contribution to relevant endpoints of toxicity .
Recently, it has been argued that grouping criteria should focus on common adverse outcomes . This approach could allow the use of shared effect biomarkers, even unspecific, since when used in combination it may reflect specific patterns of biochemical changes (signatures) induced by a mixture of chemicals. However to date, most of the outcomes pertaining to Pb, As and Mn biomarkers have relied on single exposure studies, and they do not permit extrapolation to their combined effects in humans (see Table 1).
With respect to biomarkers of effect induced by metal mixtures, even less progress has been achieved , despite the knowledge on interactions between metals at numerous biological levels . Interactions between metals may also result in altered toxicokinetics and toxicodynamic processes . Added effects of Pb and As on the monoaminergic systems in have been shown . Depressed levels of DA were found in the brain of rats co-exposed to Pb and Mn . Changes in the dopaminergic marker serum PRL in children were associated with the environmental exposure to a mixture of four metals that included Pb, As and Mn . Concomitantly the experimental co-exposure to Pb and As caused synergistic inhibition of ALAD and increased ALA urinary excretion as compared to a single exposure either to Pb or As [143, 144].
The Pb, As and Mn mixture is present in several environmental, occupational and food contexts. This mixture may induce synergetic or added toxic effects to those inherent to single metal exposure, rendering the risk of increased toxicity a likely scenario. The selection of biomarkers based on analogous endpoints of toxicity should be a useful tool for predicting and preventing the risk of toxicity. Induction of oxidative stress, interference with cholinergic or dopaminergic systems and altered heme synthesis are shared mechanisms of Pb, - As- and Mn-induced toxicity. A better understanding on the interactive toxic mechanisms is necessary to further delineate a rational basis for the their biomarkers of effect, thus enhancing the tool-kit available for risk assessment, and ultimately limiting exposure and preventing health risks in exposed populations.
The authors wish to acknowledge financial support from FCT strategic project PEst-OE/SAU/UI4013/2011, Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa and from the National Institute of Health (NIH R01ES10563).