Biomarkers of Lead Exposure and DNA Methylation within Retrotransposons

Wright, Robert O., Schwartz, Joel, Wright, Rosalind J., Bollati, Valentina, Tarantini, Letizia, Park, Sung Kyun, Hu, Howard, Sparrow, David, Vokonas, Pantel, Baccarelli, Andrea, Environmental Health Perspectives

Background: DNA methylation is an epigenetic mark that regulates gene expression. Changes in DNA methylation within white blood cells may result from cumulative exposure to environmental metals such as lead. Bone lead, a marker of cumulative exposure, may therefore better predict DNA methylation than does blood lead.

OBJECTIVE: In this study we compared associations between lead biomarkers and DNA methylation.

METHODS: We measured global methylation in participants of the Normative Aging Study (all men) who had archived DNA samples. We measured patella and tibia lead levels by K-X Ray fluorescence and blood lead by atomic absorption spectrophotometry. DNA samples from blood were used to determine global methylation averages within CpG islands of long interspersed nuclear elements-1 (LINE-1.) and Alu retrotransposons. A mixed-effects model using repeated measures of Alu or LINE-1 as the dependent variable and blood/bone lead (tibia or patella in separate models) as the primary exposure marker was fit to the data.

RESULTS: Overall mean global methylation ([+ or -] SD) was 26.3 [+ or -] 1.0 as measured by Alu and 76.8 [+ or -] 1.9 as measured by LINE-1. In the mixed-effects model, patella lead levels were inversely associated with LINE-1 [beta] = -0.25;p < 0.01) but not Alu ([beta] = -0.03; p = 0.4). Tibia lead and blood lead did not predict global methylation for either Alu or LINE-1.

CONCLUSION: Patella lead levels predicted reduced global DNA methylation within LINE-1 elements. The association between lead exposure and LINE-1 DNA methylation may have implications for the mechanisms of action of lead on health outcomes, and also suggests that changes in DNA methylation may represent a biomarker of past lead exposure.

KEYWORDS: aging, DNA methylation, epigenetics, lead, metals. Environ Health Perspect 118:790-795 (2010). doi:10.1289/ehp.090l429 [Online 11 January 2010]

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Epigenetics can be defined broadly as the study of the temporal and spatial regulation of gene expression. Changes in epigenetic marks can be as profound as DNA sequence mutations, but unlike DNA mutations, epigenetic marks are reversible and responsive to the environment (Baccarelli and Bollati 2009). DNA methylation is the best studied of the epigenetic processes that regulate gene expression. DNA methylation typically occurs in cytosines within CpG repeat sequences found in gene promoter regions. Areas enriched in CpG repeats are frequently referred to as "CpG islands." Methylation of DNA within promoter regions regulates local gene transcription. In general, increased DNA methylation is inversely associated with gene expression. Much epigenetic research has been focused on the heritability of phenotypic traits not attributable to alterations in DNA sequence, but such epigenetic modifications would have to occur in germ cells to be truly heritable. Changes in epigenetic marks in somatic cells are also of great interest, as these changes can also be induced by environmental exposures and may play a role in the toxicity of environmental agents, even if they are not heritable.

Previous research in both animals and humans has noted that DNA methylation can be altered by exposure to environmental metals (Reichard et al. 2007; Sciandrello et al. 2004; Takiguchi et al. 2003). Oxidative stress may be a unifying process to explain these findings across different metals. Metals are known to increase production of reactive oxygen species in a catalytic fashion via redox cycling (Fowler et al. 2004; Valko et al. 2005). Oxidative DNA damage can interfere with the ability of methyltransferases to interact with DNA (Turk et al. 1995; Valinluck et al. 2004), thus resulting in a generalized hypo-methylation of cytosine residues at CpG sites (Turk et al. 1995). Long-term exposure to oxidative stress has been shown to result in oxidative damage of methylated cytosine residues and depletion in the level of 5-methyl-cytosine in repeated elements (Pogribny et al. 2007; Valinluck et al. 2004). Lead is among the most prevalent of toxic environmental metals, has substantial oxidative properties, and could also alter epigenetic marks after long-term exposure.

DNA methylation can be measured both in specific genes and using global DNA methylation markers such as repetitive DNA sequences (Bollati et al. 2007; Kazazian and Moran 1998; Pilsner et al. 2007; Roman-Gomez 2005). Measures of global methylation typically involve methylation within repetitive DNA sequence. The two primary types of repetitive elements studied are Alu and long interspersed nucleotide elements (LINE-1) (Bollati et al. 2007; Issa 2002; Pilsner et al. 2007). There are approximately 1.4 million Alu repetitive elements in the human genome and half a million LINE-1 elements. LINE-1 and Alu are sometimes referred to as "junk" DNA. They are remnants of viral DNA (transposons) or viral RNA (retrotransposons) that were incorporated into the human genome over evolutionary history (Carnell and Goodman 2003; Wallace et al. 2008a, 2008b). If transcribed, such elements can reinsert into DNA, potentially moving across the genome. These elements are rich in CpG repeats and are typically highly methylated in order to suppress their expression (Schulz et al. 2006). Because such elements represent approximately half of all DNA sequences, changes in methylation in LINE-1 and Alu are believed to be indicative of global or genomic methylation (Bestor and Tycko 1996; Klose and Bird 2006).

In this study, we analyzed global DNA methylation markers (Alu and LINE-1) in a cohort of elderly men who have been well characterized for their environmental cumulative exposure to lead. Because of the stable, yet modifiable nature of DNA methylation, we hypothesized that bone lead, a marker of cumulative exposure with a half-life measured in years, would be associated with changes in global DNA methylation and that this association would be more evident than an association with a biomarker of concurrent/ short-term exposure such as blood lead.

Methods

This study was approved by the institutional review boards of Harvard School of Public Health, Brigham and Women's Hospital, and the Boston Veteran's Administration Hospital, and all participants gave written informed consent. This study was conducted on a subsample of the Normative Aging Study (NAS), a multidisciplinary longitudinal study of aging established by the Veterans Administration in 1963 (Bell et al. 1966). Briefly, 2,280 men were enrolled in the NAS. Participants received their first medical examination between 1963 and 1969. Subsequently, subjects have reported for medical examinations and standard blood and urine tests every 3-5 years. During those visits, NAS participants filled out questionnaires on smoking history, education level, food intake, and other risk factors that may influence health. Beginning in 1991, those who gave their informed consent presented to the Ambulatory Clinical Research Center of Brigham and Women's Hospital for a K-X-ray fluorescence (KXRF) measurement of lead content in the tibia and patella. Study subjects were thus measured for bone lead between 1991 and 1999.

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Publication information: Article title: Biomarkers of Lead Exposure and DNA Methylation within Retrotransposons. Contributors: Wright, Robert O. - Author, Schwartz, Joel - Author, Wright, Rosalind J. - Author, Bollati, Valentina - Author, Tarantini, Letizia - Author, Park, Sung Kyun - Author, Hu, Howard - Author, Sparrow, David - Author, Vokonas, Pantel - Author, Baccarelli, Andrea - Author. Journal title: Environmental Health Perspectives. Volume: 118. Issue: 6 Publication date: June 2010. Page number: 790+. © 2006 National Institute of Environmental Health Sciences. COPYRIGHT 2010 Gale Group.

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