Research Biomarkers of Lead Exposure and DNA Methylation within Retrotransposons Robert O. Wright,1,2 Joel Schwartz,1 Rosalind J. Wright,1,3 Valentina Bollati,4 Letizia Tarantini,4 Sung Kyun Park,5 Howard Hu,5 David Sparrow,6 Pantel Vokonas,6 and Andrea Baccarelli 1,4 1Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts, USA; 2Department of Pediatrics, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA; 3The Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; 4Laboratory of Environmental Epigenetics, Department of Occupational and Environmental Health, IRCCS Maggiore Policlinico Hospital, Mangiagalli e Regina Elena Foundation and Università degli Studi di Milano, Milan, Italy; 5Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan, USA; 6VA Normative Aging Study, VA Boston Healthcare System and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA 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. o Bvitcej : In this study we compared associations between lead biomarkers and DNA methylation. Msdohte : 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. r tluse : Overall mean global methylation (± SD) was 26.3 ± 1.0 as measured by Alu and 76.8 ± 1.9 as measured by LINE-1. In the mixed-effects model, patella lead levels were inversely associated with LINE-1 (β = –0.25; p < 0.01) but not Alu (β = –0.03; p = 0.4). Tibia lead and blood lead did not pre- dict global methylation for either Alu or LINE-1. c onclusion : Patella lead levels predicted reduced global DNA methylation within LINE-1 elements. The association between lead exposure and LINE-1 DNA methylation may have implica- tions 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. k ye sdrow : aging, DNA methylation, epigenetics, lead, metals. Environ Health Perspect 118:790–795 (2010). doi:10.1289/ehp.0901429 [Online 11 January 2010] 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 environ­ ment (Baccarelli and Bollati 2009). DNA methylation is the best studied of the epige­ netic processes that regulate gene expression. DNA methylation typically occurs in cyto­ sines 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 pro­ moter regions regulates local gene transcrip­ tion. 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 environ­ mental agents, even if they are not heritable. Previous research in both animals and humans has noted that DNA methylation 790 can be altered by exposure to environmen­ tal 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). Long­term exposure to oxidative stress has been shown to result in oxidative damage of methylated cytosine resi­ dues and depletion in the level of 5­methyl ­ 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 (LINE­1) (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 LINE1­ elements. LINE1­ and Alu are sometimes referred to as “junk” DNA. They are remnants of viral DNA (transposons) or viral RNA (retrotranspo­ sons) 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 LINE1­ 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 LINE­1) in a cohort of elderly men who have been well characterized for their environmental cumula­ tive 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 A d d r e s s c o r r e s p o n d e n c e t o R . O . W r i g h t , Department of Environmental Health, Harvard School of Public Health, Landmark Center, Third Floor East, 401 Park Dr., Boston, MA 02215 USA. Telephone: (617) 384­ 8870. Fax: (617) 384­ 8994. E­mail: rowright@hsph.harvard.edu The Normative Aging Study is supported by the Cooperative Studies Program/Epidemiology Research and Information Center of the U.S. Department of Veterans Affairs and is a compo­ nent of the Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC), Boston, MA, USA. This study was also supported by grants R01ES015172, ES014663, R01ES005257, R01ES013744, K23ES000381, and P30ES00002 from the National Institutes of Health; and by the Leaves of Grass Foundation, Newton, MA, USA. The authors declare they have no actual or poten­ tial competing financial interests. Received 6 September 2009; accepted 11 January 2010. ovlume 118 | number 6 | June 2010 • Environmental Health Perspectives