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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Biol Trace Elem Res. 2014 Apr 9;158(3):384–391. doi: 10.1007/s12011-014-9954-2

Arsenic and Manganese Alter Lead Deposition in the Rat

V Andrade a, ML Mateus a, D Santos a, M Aschner b, MC Batoreu a, AP Marreilha dos Santos a,*
PMCID: PMC4041197  NIHMSID: NIHMS584292  PMID: 24715659

Abstract

Lead (Pb) continues to be a major toxic metal in the environment. Pb exposure frequently occurs in the presence of other metals, such as arsenic (As) and manganese (Mn). Continued exposure to low levels of these metals may lead to long-term toxic effects due to their accumulation in several organs. Despite the recognition that metals in a mixture may alter each other’s toxicity by affecting deposition, there is dearth of information on their interactions in vivo. In this work, we investigated the effect of As and Mn on Pb tissue deposition, focusing on the kidney, brain and liver.

Wistar rats were treated with 8 doses of each single metal, Pb (5 mg/Kg bw), As (60 mg/L) and Mn mg/Kg bw), or the same doses in a triple metal mixture. Kidney, brain, liver, blood and urine Pb, As and Mn concentrations were determined by graphite furnace atomic absorption spectrophotometry.

Pb kidney, brain and liver concentrations in the metal mixture-treated group were significantly increased compared to the Pb alone treated group, being more pronounced in the kidney (5.4 fold), brain (2.5 fold) and liver (1.6 fold). Urinary excretion of Pb in the metal mixture-treated rats significantly increased compared with the Pb treated group, although blood Pb concentrations were analogous to the Pb treated group.

Co-treatment with As, Mn and Pb alters Pb deposition compared to Pb alone treatment, increasing Pb accumulation predominantly in kidney and brain. Blood Pb levels, unlike urine, do not reflect the increased Pb deposition in the kidney and brain. Taken together, the results suggest that the nephro- and neurotoxicity of “real-life” Pb exposure scenarios should be considered within the context of metal mixture exposures.

Keywords: Lead, arsenic, manganese, metal mixtures, brain and kidney deposition

1. Introduction

Human exposure to metals and semi-metals persists, given their emission by industries [1]. Among them, lead (Pb), arsenic (As) and manganese (Mn) are of considerable concern to environmental and public health issues [2], owing to their inherent toxicity [3]. While Pb and Mn are metals As is a semi-metal, but aiming simplification purposes along the text, all the three elements will be referred as metals. Pb and As are present in the top ten binary combinations of contaminants in soil and water [4] with major occupational exposures reported in metal smelters [5, 6]. Co-exposures to Pb and Mn have been described in children as well as in several occupational cohorts [7]. The 3 metals are among the major toxicants in mining environments [3], with concurrent exposures from soil and dust [8, 9].

Continued exposure to Pb, As and Mn may lead to long-term toxic effects [3], since these metals readily accumulate in multiple tissues [10]. Food- and water-borne Pb is distributed to several organs after gastrointestinal (GI) absorption [11, 12, 13, 14] where 3–10% (for adults) of an oral dose of water-soluble Pb can be absorbed [12] and can induce neurological, hematological and nephrotoxic effects [13, 15]. Unabsorbed Pb is excreted predominantly in the feces and absorbed Pb is excreted mainly in the urine [11]. Approximately 80–90% of As is absorbed from the GI tract [11]; skin and lungs are also considered relevant routes of occupational exposure [11]. Arsenic predominantly deposits in the liver [16], kidney and brain [17], triggering nephro- and neurological effects [18]; its predominant route of excretion is via urine [19]. Although Mn is an essential element, exposure to high levels may result in toxic effects [20]. Inhalation is the predominant route of exposure to Mn in occupational cohorts [10% absorbed by the lungs, [21] although GI absorption (less than 5 %) [11] may also happen in occupational contexts. Mn readily deposits in brain, with half-lives exceeding those in other organs [7, 20]. Upon ingestion, Mn is widely distributed via plasma, accumulating in mitochondria-rich tissues, such as brain, liver and kidney. High Mn levels adversely affect brain and liver function [20]. Mn is predominantly eliminated by fecal excretion [11].

The absorption, distribution, metabolism and excretion (ADME) of Pb, As and Mn is reasonably well understood but only for single metal exposure. Despite the general recognition that each mixture component may affect the toxicokinetics of the other mixture components [22], there is a dearth of information on the in vivo interaction between metals [23].

Pb and As are non-essential metals for biological function [18, 24]. In contrast, given its essentiality, a range of biochemical mechanisms (membrane transporters) regulate Mn’s uptake and homeostasis [25]. Essential metal transporters can be “hijacked” by nonessential metals possessing physico-chemical similarities [10]. Accordingly, competition between metals within a mixture may take place at relevant surface transporters, modifying their uptake and accumulation [26]. In addition, once within the cells, a particular metal may occupy abundant binding sites [27] on metalloproteins or target molecules, in turn, modifying the compartmentalization of other metals, thus leading to aberrant binding and toxicity [26]. A given metal may induce overexpression of transporters and/or binding proteins that alter the uptake of other metals [27, 28]. These events may modify the uptake and/or retention of metals in cells, affecting their toxicokinetics profile and toxicity [1, 29].

Humans are more frequently exposed to toxic mixtures rather than to a single element. Therefore, it is essential to understand how chronic low-level co-exposures modify metal deposition and toxicity [1]. Previous work from our group has demonstrated that rats co-treated a mixture of Pb, As and Mn show behavioral motor dysfunction compared to animals treated with each single metal alone [30]. The present study was designed to test the hypothesis that tissue Pb levels are modified by co-treatments with As and Mn, accounting for the exacerbated behavioral deficits previously ascertained in the metal mixture-treated rats. Therefore, an in vivo study with rats repeatedly exposed to Pb, As and Mn alone, and also to a mixture of these metals, was conducted to determine changes in Pb levels in various organs and fluids.

2. Materials and Methods

2.1 Chemicals

Manganese (Mn) standard for Graphite Furnace Atomic Absorption Spectrometry (GFAAS) from Fluka; arsenic (As) standard solution (H3AsO4), di-sodium hydrogen phosphate p.a. (Na2HPO4; ≥ 99%), hydrogen peroxide 30% (H2O2), magnesium matrix modifier for GFAAS [Mg(NO3)2 ・ 6H2O], nitric acid 65% suprapure (HNO3) from Merck; lead acetate trihydrate puriss. [Pb(CH3CO2)2·H2O], lead for AAS standard solution, manganese chloride tetrahydrate (MnCl2·4H2O; 99.99%), sodium (meta) arsenite purum (AsO2Na; ≥ 99%) from Sigma.

2.2 In vivo assay

An in vivo assay with male Wistar rats repeatedly exposed for 8 days was performed. The rats (weighing 165 –206 g) were purchased from Charles River Laboratories®, Barcelona. All experiments were carried out in accordance with criteria outlined in the guiding principles of the European Community Council Directive (89/609/EEC) for the care and use of laboratory animals. The rats were housed in a room with controlled temperature, humidity and a 12-h light/dark cycle. General health was checked on a daily basis. All animals had free access to water and food, supplied as pellets and adequate for normal growth and maintenance.

After an acclimatization period of 15 days, the animals were randomly assigned to 5 groups, 6 rats per group. Treatments were performed during 8 days according with the following protocol: group 1 (control group), a sterile saline solution was administrated via intraperitoneal (i.p.); group 2 (As) was exposed to 60 mg AsO2Na/L via drinking water; group 3 (Mn) 10 mg MnCl2/kg bw was administrated via i.p.; group 4, 5 mg C4H6O4 Pb/kg bw was administrated via i.p.; group 5 (Mixt) was exposed to the mixture of these 3 metals (As, Mn and Pb) with the same doses and conditions of each single exposed group.

Urine (24-h) was collected in metabolic cages before the beginning of the treatment, and after the last dose. Prior to sacrifice (24 h after the last dose), rats were anesthetized with pentobarbital (20 mg/kg, i.p.), blood was collected by cardiac puncture, and liver, kidney and brains were immediately dissected out on ice and stored at −80°.

2.3. Analyses of Pb, As and Mn

I- Sample preparation: a- Urine (2.5 mL) was digested with 1.25 mL of an acid mixture of 1:1 (v/v) 65% HNO3: HCl at 100°C for 30 minutes, in a water bath; b- 80 mg of renal cortex (after removing the external kidney membranes) was dried-out for 5 hours at 90°C prior to digestion with 1.5 mL of 65% HNO3 at 100°C for 6 hours in a dry heater block; c- 100 mg of blood and 80 mg of liver or brain samples were digested by Microwave-Assisted acid digestion (900 W, 30”) using Parr Microwave Acid Digestion Bombs® with 2.9 mL of oxidizing acid mixture containing 4:1 (v/v) HNO3 65% suprapure: H2O2 30%. The acid digested solutions were transferred to volumetric flasks (25 mL) and the volume supplemented with deionized water and kept at 4°C until analysis.

II- Atomic absorption spectrophotometry analysis: Brain and urinary Pb, As and Mn concentrations were determined by graphite furnace atomic absorption spectrophotometry GFAAS (PerkinElmer AAnalyst™ 700) equipped with an HGA, a programmable sample dispenser (AS 800 Auto Sampler) and WinLab for AA software. Daily calibration curves for each element were obtained with standard solutions of Pb, As and Mn. Mg (NO3)2 was used as chemical modifier and added to blanks, standards and samples in equal volumes. The quantification limits (QLs) were 3.8 µgPb/L, 22.5 µg As/L and 5.2 µg Mn/L.

Results were expressed as µg Pb, As or Mn per g tissue (for blood, liver and kidney), per g brain protein, and as mg per g of urinary creatinine. Urinary levels of creatinine were determined by a colorimetric method with a Randox (CR510) commercial kit and brain protein contents according to Bradford protein assay (1976)[31].

2.4. Statistical Analysis

Statistical analysis was performed with the SPSS 16.0 statistical package for Windows (SPSS, Inc., Chicago, IL, USA). Data are expressed as means ± standard deviations (SD). All parameters were compared by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test to assess differences between groups. Two-way ANOVA was also performed to test for interactions between Pb, As and Mn. The significance of the results was considered when p values were less than 0.05.

3. Results

Pb, As and Mn concentrations were determined in the blood. All the 3 metals were significantly (p<0.05) increased in the respective single metal treated group as well as in the metal mixture-treated rats compared to all the other groups, including controls (Fig. 1 A, B and C).

Figs. 1.

Figs. 1

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A, B and C- Blood levels of Pb (A), As (B) and Mn (C) in control (S), As, Mn, Pb and mixture (As/Mn/Pb) treated groups (n=6 each group). In Fig 1 B, arsenic (As) levels in control, Mn- and Pb-treated groups were below the detection limit (1). Data represent the mean ± SD. All groups were compared by ANOVA and post hoc Tukey’s tests: *, ^, º, and » are p < 0.05 versus C, As, Mn and Pb.

Kidney Pb levels in the Pb- and metal mixture-treated groups were significantly (p<0.05) higher compared to all the other groups. In addition, Pb concentrations in the metal mixture-treated animals were significantly higher than the Pb alone treated group (p<0.05)(Fig. 2 A).

Figs. 2.

Figs. 2

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A, B and C- Kidney levels of Pb (A), As (B) and Mn (C) in control (S), As, Mn, Pb and mixture (As/Mn/Pb) treated groups (n=6 each group). In Fig. 2 B, arsenic (As) levels in control, Mn- and Pb-treated groups were below the detection limit (1). Data represent the mean ± SD. All groups were compared by ANOVA and post hoc Tukey’s tests: *, ^, º, and » are p < 0.05 versus C, As, Mn and Pb

Kidney As concentrations were analogous in the As- and the metal mixture-treated groups and significantly higher compared to the other groups (p<0.05)(Fig. 2 B). Kidney Mn levels were significantly (p<0.05) higher in the Mn- and metal mixture-treated groups compared to the control, As-, and Pb-treated groups (Fig. 2 C). In addition, kidney Mn concentrations in the As- and Pb-treated rats were significantly higher than in the controls (p<0.05).

Brain Pb levels were significantly higher in the Pb- and metal mixture-treated animals compared to all the other groups (p<0.05). Additionally, brain Pb levels in the metal mixture-treated group were significantly higher than in the Pb alone treated rats (p<0.05) (Fig. 3 A). Brain As levels in the As- and metal mixture-treated rats were significantly higher compared to all the other groups (p<0.05) (Fig.3 B). Brain Mn levels were significantly higher (p<0.05) in the Mn- and metal mixture-treated animals compared to control As-, and Pb-treated groups (p<0.05) (Fig. 3 C).Kidney Pb levels in the Pb- and metal mixture-treated groups were significantly higher compared to all the other groups. In addition, Pb concentrations in the metal mixture-treated animals were significantly higher than the Pb alone treated group (p<0.05)(Fig. 2 A).

Figs. 3.

Figs. 3

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A, B and C- Brain levels of Pb (A), As (B) and Mn (C) in control (S), As, Mn, Pb and mixture (As/Mn/Pb) treated groups (n=6 each group). In Fig. 3 B arsenic (As) levels in control, Mn- and Pb-treated rats were below the detection limit (1). Data represent the mean ± SD. All groups were compared by ANOVA and post hoc Tukey’s tests: *, ^, º, and » are p < 0.05 versus C, As, Mn and Pb.

Liver Pb concentrations were significantly (p<0.05) higher in the Pb- and mixture-treated rats compared to all the other groups (Fig. 4 A). Moreover, liver Pb levels in the mixture-treated group were significantly (p<0.05) higher than in the Pb alone treated group (p<0.05) (Fig. 4 A). As liver concentrations in the As- and in the metal mixture treated rats were significantly higher than in all the other groups, although they analogous to each other (p<0.05) (Fig. 4 B).

Figs. 4.

Figs. 4

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A, B and C- Liver levels of Pb (A), As (B) and Mn (C) in control (S), As, Mn, Pb and mixture (As/Mn/Pb) treated groups (n=6 each group). In Fig. 4 B arsenic (As) levels in control, Mn- and Pb-treated rats were below the detection limit (1). Data represent the mean ± SD. All groups were compared by ANOVA and post hoc Tukey’s tests: *, ^, º, and » are p < 0.05 versus C, As, Mn and Pb.

Liver Mn concentrations in the metal mixture-treated group were significantly (p<0.05) higher compared to the other groups, except the Mn-treated group. In fact, with respect to Mn treated rats the augment of the co-treated group was small and lacked statistical significance (Fig. 4 C).

Fig. 5 A depicts that in the group treated with Pb alone, Pb urinary levels were significantly (p<0.05) higher compared to the Mn-treated and control groups (Fig. 5 A). In addition urinary Pb levels in the metal mixture-treated rats were significantly (p<0.05) higher compared to all the other groups. Urinary As levels in the As- and metal mixture-treated rats were significantly higher compared to the other groups (p<0.05) (Fig. 5B). Urinary Mn concentrations in the metal mixture-treated rats were significantly higher compared to the other groups (p<0.05) (Fig. 5 C), except the Pb-treated group. No significant differences were noted for urinary Mn levels between the single metal-treated groups and controls.

Figs. 5.

Figs. 5

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A, B and C- Urinary levels of Pb (A), As (B) and Mn (C) in control (S), As, Mn, Pb and mixture (As/Mn/Pb) treated groups (n=6 each group). In Fig. 5 B arsenic (As) levels in control, Mn- and Pb-treated groups were below the detection limit (1). Data represent the mean ± SD. All groups were compared by ANOVA and post hoc Tukey’s tests: *, ^, º, and » are p < 0.versus C, As, Mn and Pb

Two-way ANOVA was performed to access interactions between Mn and Pb and between As and Pb in the blood, liver, kidney and brain of the mixture treated group. Statistically significant (p<0.05) interactions were found in all the tissues, except between Mn and Pb in the blood and As and Pb in the kidney.

4. Discussion

Multi-metal exposure is ubiquitous, necessitating the assessment of potential combined health risks [1, 32]. Indeed, the synergistic or additive effects of metal mixtures may result in modulation of total metal tissue burden, as well as changes in metal compartmentalization [26]. Given that Pb is a persistent toxic agent and co-exists in environmental settings with As and Mn [8, 9, 33], the objective of the present study was to assess the in vivo interactions between As and Mn with Pb, focusing on changes in tissue deposition. We hypothesized that interactions between As, Mn and Pb would alter metal deposition in select target organs. Our work revealed that metal mixture treatment increased Pb concentrations, exceeding those in Pb alone treated animals (Figs. 2, 3 and 4). Given that simultaneous exposures to these metals occur in “real-life” scenarios, synergistic or additive toxic effects of these metals must to be carefully considered.

Blood

In the metal mixture-treated group, blood concentrations of the 3 metals were significantly (p<0.05) higher compared to controls, but similar to the respective, single metal-treated groups (Figs. 1A, B and C). It appears that no interactions took place between these metals during absorption or distribution, otherwise differences in their blood levels would have likely been detected. Over 90% of Pb and a great part of Mn in the bloodstream is bound to red blood cells (RBC) [34, 35] with Ca2+ channels and DMT1 implicated in their uptake [36, 37, 38]. Mn uptake occurs predominantly by non-saturable passive diffusion [39]. Within the RBCs, Pb primarily binds to delta-aminolevulinic acid dehydratase (ALA-D) (which is a saturable substrate) and secondarily to hemoglobin, while Mn binds predominantly to hemoglobin [34, 35, 39]. We think that the administered doses were insufficient for triggering competition between Pb and Mn for hemoglobin binding in the blood (Figs. 1A and C); since most of the present Pb is likely bonded to ALA-D, we posit that hemoglobin remains available for Mn binding. RBCs also take up As, although this metal enters these cells predominantly via the glucose transporter GLUT1 [40]. Once inside the RBCs, As binds to hemoglobin [41] and to ALAD [42].

Target organs

Liver, kidney and Pb levels in the metal mixture-treated group increased significantly (p<0.05) compared to controls as well as to all the single metal-treated groups (Figs. 2A, 3A and 4A). Thus, exposure to the metal mixture seemed to increase the availability of Pb. Pb content in the metal mixture-treated rats was highest in the kidney (5.4 fold), followed by the brain (2.5 fold); the liver showed the lowest increase (1.6 fold) compared to the Pb alone treated group (Figs. 2A, 3A and 4A). A dearth of information exists on Pb deposition within the context of exposures to metal mixtures. As previously observed in other works [6], it has been noted in our experiment that the co-administration of Pb and As increases brain Pb levels, and decreases As levels compared to single metal treatments. The co-administration of Pb and Mn in rats has also been shown to increase brain and kidney Pb levels, and to decrease liver Pb concentrations compared to animals treated with Pb alone, corroborating other author’s studies [27, 43]. Decreased Mn excretion in Pb pre-treated rats was posited to account for increased levels of Mn in brain, blood, lungs, kidneys and testes in co-treated animals [28]. Several putative mechanisms regulate Pb uptake, such as DMT1 [10], Ca2+ channels [44] and anion exchangers [45]. By its turn DMT1 as well as Ca2+ channels also transport Mn2+ [10, 46]. These transporters are present in the kidney, at the BBB endothelium and both glia and neurons [10, 46, 47, 48]. In this study, statistically significant (p<0.05) interaction were noted between Pb and Mn levels in the kidney and brain; In both organs, there was a significant (p<0.05) increase of Pb deposition in the mixture treated group as compared with the Pb single exposed group, while a decrease of Mn in the rats treated with the mixture was attained, when the group was compared with the group only exposed to Mn (Figs. 2A and C and 3A and C) likely due to competition between these metals.

Intraneuronal Pb2+ can functionally displace Ca2+ by impeding its entry through membrane voltage-sensitive channels; Notably, Ca2+ depletion itself can increase Pb2+ uptake [49], suggesting that Pbinduced Ca2+-depletion may increase the retention of Pb (Fig.2A and 3A). Per DMT1, no evidence exists in support of greater affinity for Pb vs. Mn for this transporter. Nevertheless, significant elevation in brain Pb levels appears to be influenced by DMT1 up-regulation secondary to changes in Mn and consequently in Fe levels [28]. The higher brain Pb levels in the metal mixture-treated group are in agreement with our observation on decreased motor function in the metal mixture-treated rats compared to single metal treated animals [30].

Intracellular retention of low doses of Pb is largely driven by its complexation with a group of inducible low molecular weight glycoproteins [50], which are particularly abundant in the kidney and brain [50, 51]. We posit that co-administration of Pb and Mn alter the binding of Pb to these low molecular weight proteins, accounting for increased Pb retention. We noted that the ratio of Pb kidney with Pb urinary levels in the metal mixture-treated rats was twice as high as the ratio calculated for the Pb single treated group (121 vs. 52, respectively). This higher retention of Pb in the metal mixture-treated rats´ kidney may account for the propensity of Pb to cause nephrotoxicity [52]. No alterations were seen in kidney As levels in the As-treated group compared to the metal mixture-treated rats. In contrast, in the brain significant interactions between As and Pb were noted (p<0.05), along with a significant (p<0.05) decrease in As concentrations when compared to the As alone treated group (Figs. 3A and 3B). We posit that the transport through the BBB can not explain these results since As but not Pb is transported by GLUT1 at this structure [40]. Further studies are needed to clarify other transport mechanism of these elements into brain.

Metallothioneins (MT) are zinc (Zn)-rich metal binding proteins (Zn) [53]. Studies on the ability of metals to displace Zn from MTs suggest a greater potency for Pb vs. As, with Mn having no ability for Zn displacement [54]. Thus, another plausible explanation for increased Pb tissue deposition in the metal-mixture treated group may be associated with its propensity to displace Zn from MT binding sites.

Pb levels in the liver of the metal mixture-treated group were significantly increased (p<0.05) compared to all the single metal-treated groups (Fig. 4A); Concerning Mn or As levels in this organ in the metal mixture-treated group, no significant changes were observed compared with the respective single metals treated rats (Figs. 4B and 4C). A potential mechanism for the increased Pb liver deposition in the metal mixture-treated group may be due to the ability of As and Pb to enhance liver MT levels [55, 56] with preferential binding of Pb given its higher affinity (vs. As) for the protein [54]. It has yet to be determined whether Mn-induced increased expression of hepatic DMT1, such as previously described in the brain [28], underlies the increased deposition of Pb in this tissue.

Excretion

A significant (p<0.05) increase in Pb excretion was observed in the metal mixture-treated rats compared to all the other groups (Fig. 5A). Urinary As levels in the metal-mixture groups were indistinguishable from those in the As alone treated rats (Fig. 5B). This is an interesting result, considering that urine is the predominant route for As elimination. The major fraction of excreted As is present as a glutathione (GSH) conjugate [57]. Although transport of a GSH–Pb complex has yet to be directly demonstrated, and making an analogy with mercury and As excretion [57, 58], such complex may represent a transportable form of Pb. Furthermore Pb (as well as As and Mn) can deplete GSH [59, 60]. Should GSH-Pb complexation represent a predominant route for Pb excretion, it is plausible that the markedly increased Pb kidney levels inherent to the metal mixture-treated group reflect additive or synergistic GSH depletion and attenuation of this transport modality, resulting in decreased As excretion.

5. Conclusions

Treatment of rats with a metal mixture of Pb, As and Mn modified Pb’s deposition in several organs as compared to its deposition in rats treated with Pb alone. The most marked increase of Pb levels was observed in kidney and brain. In addition, in the metal mixture-treated rats, blood Pb levels fail to reflect increased Pb deposition in kidney and brain, raising concern that blood Pb levels may underestimate risk associated with Pb-induced kidney and brain damage. Correlation between kidney and brain Pb levels in co-treated rats and selected endpoints of nephro- and neurotoxicity should be investigated to support the proposal of re-evaluation of Pb exposure limits in this metal mixture.

Acknowledgements

The authors wish to acknowledge financial support from Fundaç900E3;o para a Ciência e Tecnologia (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 ES R0110563, P30 ES000267). The authors also wish to thank Dr. Maria Eduarda Mendes for her guidance on atomic absorption spectrometry.

Footnotes

Ethics conduct in experimental procedure

All experiments were carried out in accordance with criteria outlined in the guiding principles of the European Community Council Directive (89/609/EEC) for the care and use of laboratory animals.

Conflict of interest statement

The authors declare that there are no conflicts of interests and that this publication has been approved by all co-authors.

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