Analysis of lead toxicity in human cells
© Gillis et al.; licensee BioMed Central Ltd. 2012
Received: 25 January 2012
Accepted: 27 July 2012
Published: 27 July 2012
Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity.
We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels.
The results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies.
KeywordsLead Heavy metals Cytokines Gene expression Peripheral blood mononuclear cells Zinc protoporphyrin
Lead (Pb) is a widely distributed industrial metal and it also is naturally present in the environment. It is an environmentally persistent element and a major global environmental hazard. The Centers for Disease Control (CDC) currently consider lead poisoning the leading environmental health threat to children in the US. Lead-based paint is a primary source of lead exposure and the major source of lead toxicity in children. Lead exposure also remains one of the leading causes of workplace illness .
Lead contamination mainly occurs through absorption via the respiratory and gastrointestinal systems. Approximately 30-40% of inhaled lead enters the bloodstream . Once absorbed, 99 percent of lead is retained in the blood for approximately 30-35 days and over the following 4-6 weeks it is dispersed and accumulated in other tissues – liver, renal cortex, aorta, brain, lungs, spleen, teeth and bones . The half-life of lead in brain tissue is about two years and in bones it persists for 20-30 years . Liver tissue is the largest repository of lead (33%) followed by the kidney cortex and medulla . Depending on the amount of exposure, lead can adversely affect the nervous system, kidneys, the immune system, reproductive and developmental systems and the cardiovascular system. Its toxic effects vary from subtle changes in neurocognitive function in low-level exposures to a potentially fatal encephalopathy in acute lead poisoning . Infants and young children are especially sensitive to low levels of lead which may contribute to behavioral problems, learning deficits and lowered IQ.
The molecular mechanisms of lead toxicity are still not clearly defined. The effects of lead on calcium fluxes and calcium-regulated events have been suggested as major mechanisms of lead neurotoxicity [6–8]. Lead also stimulates calmodulin and cAMP phosphodiesterase and enhances calmodulin-mediated protein phosphorylation in synaptic vesicles ( and references therein), thereby interfering with calcium/calmodulin-mediated neurotransmitter release. Another potential mechanism of lead toxicity is the ability of lead to induce oxidative stress. The deleterious effects of lead exposures can involve both the generation of reactive oxygen or nitrogen species (ROS) and a direct depletion of the antioxidant reserves (reviewed in ). Lead decreases glutathione levels by directly binding to thiol groups and inhibiting glutathione reductase . Lead also inhibits δ-aminolevulinic acid dehydrogenase (ALAD) resulting in increased levels of δ-aminolevulinic acid (ALA) which is known to stimulate ROS production . Lead also can stimulate membrane lipid peroxidation by binding to phosphatidylcholine in the cellular membrane and inducing changes in membrane biophysical properties [12, 13]. ROS production and the generation of other potentially genotoxic compounds are possible mechanisms of the carcinogenicity of lead .
The diagnosis of lead poisoning has traditionally relied on measuring blood lead and zinc protoporphyrin levels. It is commonly accepted that the blood level concentration is the single best indicator of recent lead exposure . The U.S. Centers for Disease Control established a 10μg/dL lead concentration in blood as the concern limit for exposures in children . The blood lead level rises within hours of exposure and remains elevated for several weeks thereafter . Due to lead’s short half-life time in the blood, blood lead tests cannot be used to diagnose or rule out evidence of exposure that occurred more than six weeks before testing. Zinc protoporphyrin or free erythrocyte protoporphyrin accumulates in erythrocytes as a result of the inhibition of heme synthesis . Protoporphyrin levels begin to rise when blood lead levels exceed 1.5 to 2 μM and remain elevated for several months after exposure . The protoporphyrin test is not as sensitive as the direct measurement of lead levels as other inhibitors of heme biosynthesis or iron deficiency anemia also increase protoporphyrin levels . The genetic deficiency of ferrochelatase, a heme biosynthetic enzyme may lead to inaccurate test results as well .
Measurements of lead in tissues are useful in estimating exposure doses but they do not document injurious effects from the amount of lead that was absorbed by the body. Consequently, a methodology to track and quantify the injurious effects of exposures to lead is not only desirable, it is critical in order to identify appropriate preventive and therapeutic methodologies. And because the injurious effects of lead are often not recognized until the disease has advanced, identifying subclinical effects of exposures to lead is vital for early intervention. In an attempt to identify the biological effects of exposures to lead as well as develop and test the methodology for evaluating lead's injurious consequences at a cellular level, we performed global gene expression profiling in human cells challenged to lead. In our recent studies, we have previously utilized this methodology to comprehensively assess unique responses to environmental toxins in cultured peripheral blood mononuclear cells (PBMC) [21, 22]. In this study we identified the cellular processes and pathways which were affected by exposures to lead, as well as the nature of cellular defences against lead insults.
We recruited a group of 44 subjects who did not have any known occupational exposures to lead and none had ever been diagnosed with lead-related disorders. The serum lead levels in the group were in a range from 0.1 to 5.8 μg/dL, with an average of 2.13 μg/dL. ZPP levels in the blood of the subjects ranged from 15-101 μg/dL with an average of 42.7 μg/dL. Since normal levels for blood zinc protoporphyrin are defined as being in the range of 16-36 μg/dL , a significant portion of the test subjects had elevated ZPP test results.
Gene expression profiling in PBMC challenged to lead
The cytokine expression study identified the specific impact of lead on mononuclear cell functions. To better understand how exposures to lead affect cellular processes on a molecular level and in order to identify intracellular responses to lead, we performed a genome-wide expression profiling in the PBMC of seven healthy individuals challenged to 10 μM lead acetate, the lowest concentration which affected cytokine production. Culturing cells in the presence of lead acetate for one day significantly altered the expression of 271 transcripts, of which expression of 221 transcripts changed 1.5-fold or more, including 157 downregulated transcripts and 64 upregulated transcripts. We observed elevated expressions of a number of cellular metabolic enzymes and metal-binding proteins which are important components in cellular defense responses against heavy metal insults. For example, of sixteen transcripts which were elevated 3-fold or more in lead-treated cells, twelve corresponded to seven metallothionein (MT) genes and the MT pseudogene. MT is a family of cysteine-rich, low molecular weight proteins which bind heavy metals through the thiol group of their cysteine residues . MT proteins are implicated in protecting against metal-induced toxicity as well as oxidative stress and their expression is induced by a number of stimuli, including exposures to metals, oxidative stress, glucocorticoids, hydric stress and others [29, 30].
Annotation clusters of genes affected by lead
Annotation Cluster 1
Enrichment Score: 9.7
metal ion-binding site: Divalent metal cation; cluster B
metal ion-binding site: Divalent metal cation; cluster A
IPR018064: Metallothionein, vertebrate, metal binding site
IPR000006: Metallothionein, vertebrate
IPR003019: Metallothionein superfamily, eukaryotic
Annotation Cluster 2
Enrichment Score: 5.8
GO:0046870 ~ cadmium ion binding
GO:0005507 ~ copper ion binding
Functional families of genes affected by lead
Group1. IPR000006: Metallothionein, vertebrate
Enrichment Score: 6.0
213629_x_at, 210524_x_at, 217165_x_at
metallothionein 1L (gene/pseudogene); metallothionein 1E; metallothionein 1 pseudogene 3; metallothionein 1J (pseudogene)
Group 2. GO:0004672 protein kinase activity
Enrichment Score: 1.2
WNK lysine deficient protein kinase 1; hypothetical LOC100132369
mitogen-activated protein kinase kinase kinase kinase 4
CDC42 binding protein kinase alpha (DMPK-like)
CDC-like kinase 4
NIMA (never in mitosis gene a)-related kinase 1
Residual effect of exposures to lead
Persistent changes in gene expression induced by lead
Probe Set ID
Entrez Gene ID
metallothionein 1 pseudogene 2
metallothionein 1E///metallothionein 1H///metallothionein 1M///metallothionein 1 pseudogene 2
endothelin receptor type B
aldehyde dehydrogenase 1 family, member A1
transmembrane protein 158
family with sequence similarity 70, member A
prostaglandin reductase 1
arrestin domain containing 4
aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha)
aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid)
malic enzyme 1, NADP(+)-dependent, cytosolic
solute carrier family 7, (cationic amino acid transporter, y + system) member 11
solute carrier family 12 (potassium/chloride transporters), member 8
pirin (iron-binding nuclear protein)
Cellular responses to lead in subjects with elevated blood lead levels
Annotation clusters affected by lead in individuals with high blood lead levels
Annotation Cluster 1
Enrichment Score: 4.6
GO:0010941 ~ regulation of cell death
GO:0042981 ~ regulation of apoptosis
GO:0043067 ~ regulation of programmed cell death
Annotation Cluster 2
Enrichment Score: 4.4
GO:0010629 ~ negative regulation of gene expression
GO:0016481 ~ negative regulation of transcription
GO:0010558 ~ negative regulation of macromolecule biosynthetic process
GO:0031327 ~ negative regulation of cellular biosynthetic process
GO:0009890 ~ negative regulation of biosynthetic process
GO:0045934 ~ negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
GO:0051172 ~ negative regulation of nitrogen compound metabolic process
GO:0010605 ~ negative regulation of macromolecule metabolic process
Annotation Cluster 3
Enrichment Score: 4.0
GO:0043068 ~ positive regulation of programmed cell death
GO:0010942 ~ positive regulation of cell death
GO:0043065 ~ positive regulation of apoptosis
Annotation Cluster 4
Enrichment Score: 3.8
GO:0016481 ~ negative regulation of transcription
GO:0045892 ~ negative regulation of transcription, DNA-dependent
GO:0051253 ~ negative regulation of RNA metabolic process
Physiological processes affected by lead in individuals with high blood lead levels
Nervous system development and function
8.32E-04 - 4.62E-02
Proliferation of Schwann cells
Growth of axons
3.62E-03 - 4.62E-02
6.34E-03 - 4.62E-02
Hematological system development and function
9.70E-03 - 4.81E-02
9.70E-03 - 3.61E-02
Gene expression profiling in subjects with elevated ZPP test results
Annotation clusters of genes affected in individuals with higher ZPP levels
Annotation Cluster 1
Enrichment Score: 2.8
GO:0050806 ~ positive regulation of synaptic transmission
GO:0051971 ~ positive regulation of transmission of nerve impulse
GO:0031646 ~ positive regulation of neurological system process
To determine if the cells from the subjects with elevated ZPP levels responded differently to the lead challenges, we compared gene expressions in the “high ZPP” group and the “normal ZPP” group in the cell samples challenged to lead. A total of 1230 transcripts were identified whose signals were different in the two study groups by a statistically significant value. Of those, only 275 transcripts had a ratio of mean signal values higher than 1.5. 146 transcripts in this group were also identified in control cultures at the 10% confidence level. Expressions of the majority of genes therefore varied between these two groups regardless of a lead challenge. A remaining 129 transcripts were found only in the cells challenged to lead and signified the difference in the responses to lead between the “high ZPP” group and the “normal ZPP” group (Additional file 1: Table S1). These results demonstrated that cellular responses to lead were altered in subjects with elevated blood ZPP levels.
For many chronic diseases, the specific role of environmental exposures in causing diseased phenotypes is not well understood. It is vital to determine when injurious exposures have occurred long before there is the appearance of overt clinical signs and symptoms in humans. Because these injurious effects most certainly develop in a discrete cellular manner, we applied gene expression and protein profiling to find and categorize these cellular effects. Cytokine profiling, in particular, is very important for understanding how environmental exposures affect the expression of these soluble mediators which are produced in tissues undergoing defence, growth, differentiation and repair processes . Even low-level exposures to lead impair cell-mediated immunity by upsetting the balance between Th1- and Th2- like T lymphocytes which alters cytokine expression [36–38]. The changes in proinflammatory cytokines also play a role in the neurotoxicity of lead [39–41]. We demonstrated that lead exposures resulted in a dose-dependent increase of IL-8 production by PBMC as well as enhanced the production of the proinflammatory cytokines IL-6, TNF-α and the chemokines IL-8 and MIP-1α in response to mitogens. The increase in the TNF-α level was in agreement with previous studies  and was consistent with epidemiological data on lead-exposed workers . Our observation that lead increases mitogenic activation of PBMC was in agreement with the activation of lymphocyte and leukocyte proliferation and function by lead which has been observed in earlier studies [24–27]. One mechanism by which lead may activate PBMC is by inducing PKC-dependent cell signaling cascades . Our findings demonstrated that exposure to lead strongly affects cell signaling pathways, particularly ATP binding and kinase activity.
In an attempt to find changes in gene expression following exposures to lead, we identified genes whose expressions were induced by lead and remained activated even after the lead was removed from the media. The most pronounced effect was the activation of the expression of metallothionein (MT) genes. MT gene expression has been identified and utilized as a biomarker of heavy metal exposures in a variety of biological systems [44–51] including in human cell lines  as well as in exposures to cadmium in humans . Our findings imply that expressions of metallothionein genes in human tissues also may be applied to assessing exposures to lead. Another gene whose expression was activated by lead was the endothelin receptor B (EDNRB). Although the mechanism of EDNRB regulation by lead is unknown, an increase of its expression may contribute to the hypertension observed in low-level lead exposures .
A number of metabolic enzymes were also upregulated by lead exposures, including the genes encoding aldehyde dehydrogenase, tryptophan 2,3-dioxygenase, malic enzyme, aspartate beta-hydroxylase, prostaglandin reductase 1 and two members of the aldo-keto reductase family 1, C1 and C2. All of these enzymes with the exception of tryptophan 2,3-dioxygenase and aspartate beta-hydroxylase are NAD(P) + dependent. Tryptophan 2,3-dioxygenase catalyzes the degradation of tryptophan into N-formyl-kynurenine. Tryptophan depletion and the accumulation of its metabolites induce cell cycle arrest and apoptosis of T lymphocytes, thereby suppressing T-cell activation [55, 56]. They also promote the differentiation of naive CD4+ T cells into regulatory T cells. An increase in the expression of this enzyme as a result of exposure to lead may explain the depletion of CD4+ T cells observed in exposed individuals [57–60]. An increased production of tryptophan metabolites was also implicated in neurodegenerative disorders . For example, an increase of tryptophan 2,3-dioxygenase expression was found in astrocytes of schizophrenic patients . Lead-dependent activation of the gene encoding this enzyme may contribute to the neurocognitive symptoms which are observed in lead-exposed individuals. Another possible mechanism by which exposures to lead can impair neurocognitive function is the dysregulation of neurosteroids. The Aldo-keto reductase family 1 enzymes, AKR1C1 and AKRC2 are implicated in steroid metabolism. AKRC1 inactivates progesterone by forming 20α-hydroxyprogesterone [63, 64]. Progesterone and its neuroactive metabolites have modulatory effects on brain function and influence social, cognitive, emotional and physical processes [65, 66]. Increased expression of AKR1C as a result of exposures to lead may change the levels of progesterone and its neuroactive metabolites, such as 3α,5α-THP, thereby contributing to neurocognitive disorders. Upregulation of the xenobiotic metabolizing enzymes AKR1C1 and AKRC2 caused by lead was also observed in primary normal human bronchial epithelial cells .
Among the enzymes that were upregulated in the cells challenged to lead were prostaglandin reductase 1, which inactivates leukotriene B4; 15-ketoprostaglandins which are important mediators of inflammation [68, 69]; aspartate beta-hydroxylase, which plays an important role in calcium homeostasis ; malic enzyme, a cytosolic, NADP-dependent enzyme which generates NADPH for fatty acid biosynthesis ; and a cytosolic isoform of aldehyde dehydrogenase 1, an enzyme in the major oxidative pathway of alcohol metabolism . Other activated genes included transcriptional cofactor pirin, an iron-binding nuclear protein involved in apoptosis ; two members of the amino acid/polyamine transport system, SLC12A8A  and SLC7A11 ; KIT ligand, the ligand of the tyrosine-kinase receptor encoded by the KIT locus, implicated in pigmentation  and cancer ; α-arrestin 4 ; transmembrane protein TMEM158; FAM70A and putative protein ENSG00000204134. Our findings that expressions of these genes were not transient and remained elevated even after the lead was removed from the system, demonstrate that they could be utilized as biomarkers in tests for assessing exposures to lead.
The U.S. Centers for Disease Control set a threshold for a significant exposure to lead at blood lead levels of 10 μg/dL or above which have an increased risk for both subclinical and overt effects from this substance . In particular, individuals with a blood lead level of 10 μg/dL or above are at high risk for peripheral neuropathies and/or chronic nephropathies, with the latter often triggering consequential hypertension. The analysis of the gene expressions demonstrated that cellular responses to lead were significantly altered in individuals with blood lead levels around 5 μg/dL, suggesting that doses lower than 10 μg/dL impair cellular functions. The most affected system was the nervous system and the most affected groups were the negative regulation of transcription and the positive regulation of apoptosis. The changes in gene expressions correlated well with the blood lead levels and were in agreement with previous studies in children .
Although the blood lead test provides accurate information on lead absorption in persons with brief acute exposures, it cannot be used for assessing past or chronic exposures. It is well known that exposures to lead are also associated with elevated blood ZPP levels which remain elevated for several months after the exposure. The analysis of blood chemistries in 44 subjects demonstrated that elevated blood ZPP levels correlated with higher blood ZPP content. Expressions of genes encoding proteins involved in the transmission of nerve impulses were significantly affected in the “high ZPP” group. Among the affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons (Figure 2). Therefore, the nervous system was highly affected in these individuals confirming clinical observations [31–33].
Blood ZPP levels are increased in individuals who are deficient in mineral iron or metabolism [19, 80], therefore ZPP measurements only provide indirect evidence of an exposure to lead. Nevertheless, the cellular responses to lead were changed in the subjects with the elevated blood ZPP levels which were signified by the altered gene expression profiles.
The present study defined discrete and unique responses to lead in cultured PBMC. Our findings define the injurious effects from lead exposures at the cellular level. The methodology of detecting the health effects of environmental exposures based upon individual cellular response patterns offers a starting point for assessing injurious consequences of such exposures and to developing appropriate health treatment protocols. This approach makes it possible to obtain human toxicity data which can be used to identify the potentially injurious effects of exposures to any occupational and environmental compound for which human toxicity data are not yet available. Human medical toxicology is based upon the appreciation that after target cells of a toxin are impacted, there are two primary phases of reaction. One is composed of the acute side effects, which are then followed by the chronic and residual impact of the toxin. We have been able to document both the “acute” and “residual” effects of lead on human cells based upon changes in gene expression patterns. We believe that gene expression profiling can be utilized whenever there is a desire to confirm lead poisoning and it therefore merits being an adjunct to blood lead testing in lead-exposed individuals, particularly in those in which an adverse impact needs to be ascertained.
The study was approved by the Institutional Review Board Services, Aurora, Ontario, Canada. We recruited 44 healthy volunteers from the Los Angeles, CA area. Volunteers were of both genders ranging in age between 18 and 54 and equally distributed among Caucasians, Hispanics, African Americans and Asians. The subjects signed the consent forms, completed the health questionnaire forms and underwent physical examinations. Those who had no history of known personal or occupational exposures to lead and no background of lead-related symptoms or disorders underwent blood tests to determine their plasma lead and ZPP levels. The subjects whose plasma lead levels were in the normal range below 10 μg/dL were asked to provide 50 ml of blood for experimentation.
Isolation and culture of PBMC
The blood was collected in (K3) EDTA collection tubes by venipuncture after obtaining appropriate informed consent. PBMC were isolated by Ficol gradient centrifugation as described earlier [21, 22] and were suspended at 106 cells/ml in RPMI 1640 medium supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The cells were cultured for 18 hours in three replicate plates in the presence of lead acetate at five concentrations: 0.08 μM, 0.4 μM, 2 μM, 10 μM and 50 μM either with or without 10μg/ml phytohemagglutinin (PHA-P, Sigma-Aldrich®, St. Louis, MO). Control cells were cultured in three replicate wells in the medium which did not contain lead. Lead concentrations were far below the toxicity levels observed in PBMC at concentrations above 500 μM .
Cell culture supernatants were collected and concentrations of 15 common cytokines GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, MIP-1β, MCP-1, Eotaxin, MIP-1α and RANTES were measured by using multiplex immunoassays based on Luminex xMAP™ bead array technology as described earlier [21, 22]. PBMC isolated from three donors were used for the cytokine analyses. Mean concentration values of cytokines in cultures challenged to lead acetate were normalized to those in control cultures. The normalized values at 10 μM and 50 μM lead acetate were compared to those at 0.08 μM and 0.4 μM lead acetate by pooled t-tests. The variances in the groups were confirmed by the F-test. All F-test p-values were above 0.1, except for IL-8 at 50 μM lead acetate. T-test with the Satterthwaite approximation to the degrees of freedom was used for this challenge. All t-tests assumed two-tailed distribution and the confidence level was set at 5%.
RNA isolation and gene array analysis
Total RNA was purified from PBMC samples by using Trizol reagent and RNeasy RNA purification kits (Qiagen Sciences, Valencia, CA) according to the manufacturer's instructions. Whole genome expression profiling was performed by using Human Genome U133 Plus 2.0 GeneChip® arrays (Affymetrix, Santa Clara, CA) interrogating over 47,000 transcripts from approximately 40,000 annotated human genes. All labeling reactions and hybridizations were carried out according to the Affymetrix GeneChip® eukaryotic target labeling protocol. Bound probes were detected by laser excitation of the fluorescent markers and the resultant emission spectra were obtained by using the Gene Array Scanner 3000 (Agilent Technologies, Santa Clara, CA). Data acquisition was performed using GCOS (Affymetrix GeneChip Operating Software Package). Data normalization, background correction and all subsequent statistical tests for significant differential expressions were performed by using a Partek statistical software package (Partek., Inc, St. Louis, MO). Data was normalized via a quintiles normalization and summarized using the Robust Multi-array Average (RMA) method . The ANOVA test was used to calculate the significance of the differential expression between treated and untreated samples. Raw p-values were corrected for FDR (false discovery rate) using the Benjamini and Hochberg procedure  and a cut-off level equal or less than 0.05 was applied. Cluster analysis was performed by using the DAVID functional annotation tool . Additional functional analysis was performed by using an Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA). Gene array data were deposited in the Gene Expression Omnibus database, accession number GSE37567 .
Peripheral blood mononuclear cells
The authors would like to acknowledge Aaron Bruno, Kristine Baraoidan, Juan Chen and WeiHua Wang for technical support during the course of this study.
- Needleman H: Lead poisoning. Annu Rev Med. 2004, 55: 209-222. 10.1146/annurev.med.55.091902.103653.View ArticlePubMed
- Philip AT, Gerson B: Lead poisoning–Part I. Incidence, etiology, and toxicokinetics. Clin Lab Med. 1994, 14 (2): 423-444.PubMed
- Patrick L: Lead toxicity, a review of the literature. Part 1: Exposure, evaluation, and treatment. Altern Med Rev. 2006, 11 (1): 2-22.PubMed
- Verstraeten SV, Aimo L, Oteiza PI: Aluminium and lead: molecular mechanisms of brain toxicity. Arch Toxicol. 2008, 82 (11): 789-802. 10.1007/s00204-008-0345-3.View ArticlePubMed
- Mudipalli A: Lead hepatotoxicity & potential health effects. Indian J Med Res. 2007, 126 (6): 518-527.PubMed
- Bressler J, Kim KA, Chakraborti T, Goldstein G: Molecular mechanisms of lead neurotoxicity. Neurochem Res. 1999, 24 (4): 595-600. 10.1023/A:1022596115897.View ArticlePubMed
- Marchetti C: Molecular targets of lead in brain neurotoxicity. Neurotox Res. 2003, 5 (3): 221-236. 10.1007/BF03033142.View ArticlePubMed
- Toscano CD, Guilarte TR: Lead neurotoxicity: from exposure to molecular effects. Brain Res Brain Res Rev. 2005, 49 (3): 529-554.View ArticlePubMed
- Patrick L: Lead toxicity part II: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. Altern Med Rev. 2006, 11 (2): 114-127.PubMed
- Gurer H, Ercal N: Can antioxidants be beneficial in the treatment of lead poisoning?. Free Radic Biol Med. 2000, 29 (10): 927-945. 10.1016/S0891-5849(00)00413-5.View ArticlePubMed
- Bechara EJ: Oxidative stress in acute intermittent porphyria and lead poisoning may be triggered by 5-aminolevulinic acid. Braz J Med Biol Res. 1996, 29 (7): 841-851.PubMed
- Adonaylo VN, Oteiza PI: Pb2+ promotes lipid oxidation and alterations in membrane physical properties. Toxicology. 1999, 132 (1): 19-32. 10.1016/S0300-483X(98)00134-6.View ArticlePubMed
- Adonaylo VN, Oteiza PI: Lead intoxication: antioxidant defenses and oxidative damage in rat brain. Toxicology. 1999, 135 (2–3): 77-85.View ArticlePubMed
- Roots LM: Tests available for assessing recent exposure to inorganic lead compounds and their use for screening purposes. Sci Total Environ. 1979, 11 (1): 59-68. 10.1016/0048-9697(79)90033-0.View ArticlePubMed
- Blood lead levels in young children--United States and selected states, 1996-1999. MMWR Morb Mortal Wkly Rep. 2000, 49 (50): 1133-1137.
- Rabinowitz MB, Wetherill GW, Kopple JD: Kinetic analysis of lead metabolism in healthy humans. J Clin Invest. 1976, 58 (2): 260-270. 10.1172/JCI108467.PubMed CentralView ArticlePubMed
- Labbe RF, Vreman HJ, Stevenson DK: Zinc protoporphyrin: A metabolite with a mission. Clin Chem. 1999, 45 (12): 2060-2072.PubMed
- Staudinger KC, Roth VS: Occupational lead poisoning. Am Fam Physician. 1998, 57 (4): 719-726. 731-712PubMed
- Martin CJ, Werntz CL, Ducatman AM: The interpretation of zinc protoporphyrin changes in lead intoxication: a case report and review of the literature. Occup Med (Lond). 2004, 54 (8): 587-591. 10.1093/occmed/kqh123.View Article
- Kansky A: Fluorescence microscopy test in porphyrias, photodermatoses and lead exposed persons. Arch Dermatol Forsch. 1975, 252 (4): 311-315.View ArticlePubMed
- Gillis B, Gavin IM, Arbieva Z, King ST, Jayaraman S, Prabhakar BS: Identification of human cell responses to benzene and benzene metabolites. Genomics. 2007, 90 (3): 324-333. 10.1016/j.ygeno.2007.05.003.View ArticlePubMed
- Gavin IM, Gillis B, Arbieva Z, Prabhakar BS: Identification of human cell responses to hexavalent chromium. Environ Mol Mutagen. 2007, 48 (8): 650-657. 10.1002/em.20331.View ArticlePubMed
- Kratz A, Ferraro M, Sluss PM, Lewandrowski KB: Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Laboratory reference values. N Engl J Med. 2004, 351 (15): 1548-1563. 10.1056/NEJMcpc049016.View ArticlePubMed
- Lawrence DA: Heavy metal modulation of lymphocyte activities--II. Lead, an in vitro mediator of B-cell activation. Int J Immunopharmacol. 1981, 3 (2): 153-161. 10.1016/0192-0561(81)90006-0.View ArticlePubMed
- Razani-Boroujerdi S, Edwards B, Sopori ML: Lead stimulates lymphocyte proliferation through enhanced T cell-B cell interaction. J Pharmacol Exp Ther. 1999, 288 (2): 714-719.PubMed
- Warner GL, Lawrence DA: Stimulation of murine lymphocyte responses by cations. Cell Immunol. 1986, 101 (2): 425-439. 10.1016/0008-8749(86)90155-3.View ArticlePubMed
- De Guise S, Bernier J, Lapierre P, Dufresne MM, Dubreuil P, Fournier M: Immune function of bovine leukocytes after in vitro exposure to selected heavy metals. Am J Vet Res. 2000, 61 (3): 339-344. 10.2460/ajvr.2000.61.339.View ArticlePubMed
- Coyle P, Philcox JC, Carey LC, Rofe AM: Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences. 2002, 59 (4): 627-647. 10.1007/s00018-002-8454-2.View ArticlePubMed
- Haq F, Mahoney M, Koropatnick J: Signaling events for metallothionein induction. Mutat Res. 2003, 533 (1–2): 211-226.View ArticlePubMed
- Klaassen CD, Liu J: Role of metallothionein in cadmium-induced hepatotoxicity and nephrotoxicity. Drug Metabolism Reviews. 1997, 29 (1–2): 79-102.View ArticlePubMed
- Lanphear BP, Dietrich K, Auinger P, Cox C: Cognitive deficits associated with blood lead concentrations <10 microg/dL in US children and adolescents. Public Health Rep. 2000, 115 (6): 521-529. 10.1093/phr/115.6.521.PubMed CentralView ArticlePubMed
- Canfield RL, Henderson CR, Cory-Slechta DA, Cox C, Jusko TA, Lanphear BP: Intellectual impairment in children with blood lead concentrations below 10 microg per deciliter. N Engl J Med. 2003, 348 (16): 1517-1526. 10.1056/NEJMoa022848.PubMed CentralView ArticlePubMed
- Cory-Slechta DA: Relationships between Pb-induced changes in neurotransmitter system function and behavioral toxicity. Neurotoxicology. 1997, 18 (3): 673-688.PubMed
- La P, Fernando AP, Wang Z, Salahudeen A, Yang G, Lin Q, Wright CJ, Dennery PA: Zinc protoporphyrin regulates cyclin D1 expression independent of heme oxygenase inhibition. J Biol Chem. 2009, 284 (52): 36302-36311. 10.1074/jbc.M109.031641.PubMed CentralView ArticlePubMed
- Hopkins SJ: The pathophysiological role of cytokines. Leg Med (Tokyo). 2003, 5 (Suppl 1): S45-S57.View Article
- Heo Y, Parsons PJ, Lawrence DA: Lead differentially modifies cytokine production in vitro and in vivo. Toxicol Appl Pharmacol. 1996, 138 (1): 149-157. 10.1006/taap.1996.0108.View ArticlePubMed
- Iavicoli I, Marinaccio A, Castellino N, Carelli G: Altered cytokine production in mice exposed to lead acetate. Int J Immunopathol Pharmacol. 2004, 17 (2 Suppl): 97-102.PubMed
- Iavicoli I, Carelli G, Stanek EJ, Castellino N, Calabrese EJ: Below background levels of blood lead impact cytokine levels in male and female mice. Toxicol Appl Pharmacol. 2006, 210 (1–2): 94-99.View ArticlePubMed
- Lahat N, Shapiro S, Froom P, Kristal-Boneh E, Inspector M, Miller A: Inorganic lead enhances cytokine-induced elevation of matrix metalloproteinase MMP-9 expression in glial cells. J Neuroimmunol. 2002, 132 (1–2): 123-128.View ArticlePubMed
- Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G: Inflammation-like glial response in lead-exposed immature rat brain. Toxicol Sci. 2007, 95 (1): 156-162.View ArticlePubMed
- White LD, Cory-Slechta DA, Gilbert ME, Tiffany-Castiglioni E, Zawia NH, Virgolini M, Rossi-George A, Lasley SM, Qian YC, Basha MR: New and evolving concepts in the neurotoxicology of lead. Toxicol Appl Pharmacol. 2007, 225 (1): 1-27. 10.1016/j.taap.2007.08.001.View ArticlePubMed
- Guo TL, Mudzinski SP, Lawrence DA: The heavy metal lead modulates the expression of both TNF-alpha and TNF-alpha receptors in lipopolysaccharide-activated human peripheral blood mononuclear cells. J Leukoc Biol. 1996, 59 (6): 932-939.PubMed
- Di Lorenzo L, Vacca A, Corfiati M, Lovreglio P, Soleo L: Evaluation of tumor necrosis factor-alpha and granulocyte colony-stimulating factor serum levels in lead-exposed smoker workers. Int J Immunopathol Pharmacol. 2007, 20 (2): 239-247.PubMed
- Bebianno MJ, Cravo A, Miguel C, Morais S: Metallothionein concentrations in a population of Patella aspera: variation with size. Sci Total Environ. 2003, 301 (1–3): 151-161.View ArticlePubMed
- Carvalho CD, de Araujo HSS, Fernandes MN: Hepatic metallothionein in a teleost (Prochilodus scrofa) exposed to copper at pH 4.5 and pH 8.0. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. 2004, 137 (2): 225-234. 10.1016/j.cbpc.2003.11.004.View Article
- Domouhtsidou GP, Dailianis S, Kaloyianni M, Dimitriadis VK: Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers - Combination with trace metal concentrations. Mar Pollut Bull. 2004, 48 (5–6): 572-586.View ArticlePubMed
- Geret F, Jouan A, Turpin V, Bebianno MJ, Cosson RP: Influence of metal exposure on metallothionein synthesis and lipid peroxidation in two bivalve mollusks: the oyster (Crassostrea gigas) and the mussel (Mytilus edulis). Aquatic Living Resources. 2002, 15 (1): 61-66. 10.1016/S0990-7440(01)01147-0.View Article
- Lecoeur S, Videmann B, Berny P: Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha. Environ Res. 2004, 94 (2): 184-191. 10.1016/S0013-9351(03)00069-0.View ArticlePubMed
- Lukkari T, Taavitsainen M, Soimasuo M, Oikari A, Haimi J: Biomarker responses of the earthworm Aporrectodea tuberculata to copper and zinc exposure: differences between populations with and without earlier metal exposure. Environ Pollut. 2004, 129 (3): 377-386. 10.1016/j.envpol.2003.12.008.View ArticlePubMed
- Perceval O, Pinel-Alloul B, Methot G, Couillard Y, Giguere A, Campbell PGC, Hare L: Cadmium accumulation and metallothionein synthesis in freshwater bivalves (Pyganodon grandis): relative influence of the metal exposure gradient versus limnological variability. Environ Pollut. 2002, 118 (1): 5-17. 10.1016/S0269-7491(01)00282-2.View ArticlePubMed
- Regoli F, Pellegrini D, Winston GW, Gorbi S, Giuliani S, Virno-Lamberti C, Bomdadre S: Application of biomarkers for assessing the biological impact of dredged materials in the Mediterranean: the relationship between antioxidant responses and susceptibility to oxidative stress in the red mullet (Mullus barbatus). Mar Pollut Bull. 2002, 44 (9): 912-922. 10.1016/S0025-326X(02)00120-0.View ArticlePubMed
- Shea J, Moran T, Dehn PF: A bioassay for metals utilizing a human cell line. Toxicol Vitr. 2008, 22 (4): 1025-1031. 10.1016/j.tiv.2008.02.014.View Article
- Lu J, Jin TY, Nordberg G, Nordberg M: Metallothionein gene expression in peripheral lymphocytes from cadmium-exposed workers. Cell Stress & Chaperones. 2001, 6 (2): 97-104. 10.1379/1466-1268(2001)006<0097:MGEIPL>2.0.CO;2.View Article
- Vaziri ND, Sica DA: Lead-induced hypertension: role of oxidative stress. Curr Hypertens Rep. 2004, 6 (4): 314-320. 10.1007/s11906-004-0027-3.View ArticlePubMed
- Lob S, Konigsrainer A, Rammensee HG, Opelz G, Terness P: Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees?. Nat Rev Cancer. 2009, 9 (6): 445-452. 10.1038/nrc2639.View ArticlePubMed
- Katz JB, Muller AJ, Prendergast GC: Indoleamine 2,3-dioxygenase in T-cell tolerance and tumoral immune escape. Immunol Rev. 2008, 222: 206-221. 10.1111/j.1600-065X.2008.00610.x.View ArticlePubMed
- Mishra KP: Lead exposure and its impact on immune system: a review. Toxicol In Vitro. 2009, 23 (6): 969-972. 10.1016/j.tiv.2009.06.014.View ArticlePubMed
- Fischbein A, Tsang P, Luo JC, Roboz JP, Jiang JD, Bekesi JG: Phenotypic aberrations of CD3+ and CD4+ cells and functional impairments of lymphocytes at low-level occupational exposure to lead. Clin Immunol Immunopathol. 1993, 66 (2): 163-168. 10.1006/clin.1993.1020.View ArticlePubMed
- Undeger U, Basaran N, Canpinar H, Kansu E: Immune alterations in lead-exposed workers. Toxicology. 1996, 109 (2–3): 167-172.View ArticlePubMed
- Li S, Zhengyan Z, Rong L, Hanyun C: Decrease of CD4+ T-lymphocytes in children exposed to environmental lead. Biol Trace Elem Res. 2005, 105 (1–3): 19-25.View ArticlePubMed
- Muller N, Myint AM, Schwarz MJ: The impact of neuroimmune dysregulation on neuroprotection and neurotoxicity in psychiatric disorders–relation to drug treatment. Dialogues Clin Neurosci. 2009, 11 (3): 319-332.PubMed CentralPubMed
- Miller CL, Llenos IC, Dulay JR, Barillo MM, Yolken RH, Weis S: Expression of the kynurenine pathway enzyme tryptophan 2,3-dioxygenase is increased in the frontal cortex of individuals with schizophrenia. Neurobiol Dis. 2004, 15 (3): 618-629. 10.1016/j.nbd.2003.12.015.View ArticlePubMed
- Jin Y, Duan L, Lee SH, Kloosterboer HJ, Blair IA, Penning TM: Human cytosolic hydroxysteroid dehydrogenases of the aldo-ketoreductase superfamily catalyze reduction of conjugated steroids: implications for phase I and phase II steroid hormone metabolism. J Biol Chem. 2009, 284 (15): 10013-10022. 10.1074/jbc.M809465200.PubMed CentralView ArticlePubMed
- Penning TM, Byrns MC: Steroid hormone transforming aldo-keto reductases and cancer. Ann N Y Acad Sci. 2009, 1155: 33-42. 10.1111/j.1749-6632.2009.03700.x.PubMed CentralView ArticlePubMed
- Zheng P: Neuroactive steroid regulation of neurotransmitter release in the CNS: action, mechanism and possible significance. Prog Neurobiol. 2009, 89 (2): 134-152. 10.1016/j.pneurobio.2009.07.001.View ArticlePubMed
- Frye CA: Neurosteroids' effects and mechanisms for social, cognitive, emotional, and physical functions. Psychoneuroendocrinology. 2009, 34 (Suppl 1): S143-S161.PubMed CentralView ArticlePubMed
- Glahn F, Schmidt-Heck W, Zellmer S, Guthke R, Wiese J, Golka K, Hergenroder R, Degen GH, Lehmann T, Hermes M, et al: Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors. Arch Toxicol. 2008, 82 (8): 513-524. 10.1007/s00204-008-0331-9.View ArticlePubMed
- Tai HH, Ensor CM, Tong M, Zhou H, Yan F: Prostaglandin catabolizing enzymes. Prostaglandins Other Lipid Mediat. 2002, 68–69: 483-493.View ArticlePubMed
- Murphy RC, Gijon MA: Biosynthesis and metabolism of leukotrienes. Biochem J. 2007, 405 (3): 379-395. 10.1042/BJ20070289.View ArticlePubMed
- Hong CS, Kwon SJ, Kim do H: Multiple functions of junctin and junctate, two distinct isoforms of aspartyl beta-hydroxylase. Biochem Biophys Res Commun. 2007, 362 (1): 1-4. 10.1016/j.bbrc.2007.07.166.View ArticlePubMed
- Hsieh JY, Chen SH, Hung HC: Functional roles of the tetramer organization of malic enzyme. J Biol Chem. 2009, 284 (27): 18096-18105. 10.1074/jbc.M109.005082.PubMed CentralView ArticlePubMed
- Vasiliou V, Pappa A: Polymorphisms of human aldehyde dehydrogenases. Consequences for drug metabolism and disease. Pharmacology. 2000, 61 (3): 192-198.PubMed
- Gelbman BD, Heguy A, O'Connor TP, Zabner J, Crystal RG: Upregulation of pirin expression by chronic cigarette smoking is associated with bronchial epithelial cell apoptosis. Respir Res. 2007, 8: 10-10.1186/1465-9921-8-10.PubMed CentralView ArticlePubMed
- Daigle ND, Carpentier GA, Frenette-Cotton R, Simard MG, Lefoll MH, Noel M, Caron L, Noel J, Isenring P: Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport. J Cell Physiol. 2009, 220 (3): 680-689. 10.1002/jcp.21814.View ArticlePubMed
- Kanai Y, Endou H: Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance. Curr Drug Metab. 2001, 2 (4): 339-354. 10.2174/1389200013338324.View ArticlePubMed
- Sturm RA: Molecular genetics of human pigmentation diversity. Hum Mol Genet. 2009, 18 (R1): R9-R17. 10.1093/hmg/ddp003.View ArticlePubMed
- Rapley EA, Nathanson KL: Predisposition alleles for testicular germ cell tumour. Curr Opin Genet Dev. 2010, 20: 1-6. 10.1016/j.gde.2010.01.001.View Article
- Patwari P, Chutkow WA, Cummings K, Verstraeten VL, Lammerding J, Schreiter ER, Lee RT: Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins. J Biol Chem. 2009, 284 (37): 24996-25003. 10.1074/jbc.M109.018093.PubMed CentralView ArticlePubMed
- Tian Y, Green PG, Stamova B, Hertz-Picciotto I, Pessah IN, Hansen R, Yang X, Gregg JP, Ashwood P, Jickling G, Van de Water J, Sharp FR: Correlations of gene expression with blood lead levels in children with autism compared to typically developing controls. Neurotox Res. 2011, 19 (1): 1-13. 10.1007/s12640-009-9126-x.PubMed CentralView ArticlePubMed
- Labbe RF: Clinical utility of zinc protoporphyrin. Clin Chem. 1992, 38 (11): 2167-2168.PubMed
- de la Fuente H, Portales-Perez D, Baranda L, Diaz-Barriga F, Saavedra-Alanis V, Layseca E, Gonzalez-Amaro R: Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells. Clin Exp Immunol. 2002, 129 (1): 69-77. 10.1046/j.1365-2249.2002.01885.x.PubMed CentralView ArticlePubMed
- Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003, 4 (2): 249-264. 10.1093/biostatistics/4.2.249.View ArticlePubMed
- Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society, Series B. 1995, 57: 289-300.
- DAVID functional annotation tool. ,http://david.abcc.ncifcrf.gov,
- Gene Expression Omnibus. ,http://www.ncbi.nlm.nih.gov/projects/geo/,
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.