Research Inhalable Metal-Rich Air Particles and Histone H3K4 Dimethylation and H3K9 Acetylation in a Cross-sectional Study of Steel Workers Laura Cantone,1 Francesco Nordio,1,2 Lifang Hou,3 Pietro Apostoli,4 Matteo Bonzini,5 Letizia Tarantini,1 Laura Angelici,1 Valentina Bollati,1 Antonella Zanobetti,6 Joel Schwartz,6 Pier A. Bertazzi,1 and Andrea Baccarelli 6 1Department of Environmental and Occupational Health, Università di Milano and Istituto Di Ricovero e Cura a Carattere Scientifico, and Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy; 2Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma Medical School, Parma, Italy; 3Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA; 4Department of Experimental and Applied Medicine, Occupational Medicine and Industrial Hygiene, University of Brescia, Brescia, Italy; 5Department of Clinical and Biological Sciences, University of Insubria, Varese, Italy; 6Laboratory of Environmental Epigenetics, Exposure Epidemiology and Risk Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts, USA c onclusi k ye sdrow Background : Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined. o Bjectivs : We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM. Methods : We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject. r esult : Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51). : Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic. : environmental carcinogens, epigenetics, histone modifications, metals, particulate matter. Environ Health Perspect 119:964–969 (2011). doi:10.1289/ehp.1002955 [Online 8 March 2011] Ambient and occupational exposure to inhalable particulate matter (PM) has been associated with increased risk of lung can- cer (Dockery et al. 1993; Gibb et al. 2000; Kuo et al. 1999). Epidemiologic and in vivo studies suggest that the metal components of PM may be responsible for PM health effects, including lung cancer (Chang et al. 2005; Chen and Hwang 2005; Conroy et al. 2008; Corey et al. 2006; Coyle et al. 2006; Franklin et al. 2008; MacNee and Donaldson 2003; Roller 2009; Wild et al. 2009). Despite the well-recognized carcinogenic potentials of sev- eral toxic metals, the molecular mechanisms underlying their associations with cancer risk remain poorly understood. In particular, most carcinogenic metals are weak mutagens and do not induce DNA adduct formation, a key initiating event caused by other carcinogens (Salnikow and Zhitkovich 2008). Growing evidence indicates that epigenetic dysregulation of gene expression plays a pri- mary role in cancer etiology (Feinberg and Tycko 2004; Ke et al. 2006). Several toxic metals have been shown to bind more avidly 964 to histone proteins than to other biopolymers such as DNA or RNA (Conroy et al. 2008; Zoroddu et al. 2000, 2002). Recent in vitro studies have shown that carcinogenic metals cause posttranslational epigenetic modifica- tions of histone proteins, thus derailing the normal programming of gene expression (Ke et al. 2006; Yan and Boyd 2006; Yan et al. 2003; Zhou X et al. 2009). In lung epithelial cell lines and malignant transformation mod- els, arsenic (Jensen et al. 2009; Zhou X et al. 2009), nickel (Zhou QX et al. 2009), and chromium (Sun et al. 2009; Zhou X et al. 2009) have been linked with specific activat- ing histone modifications, such as H3K4 (his- tone 3 lysine 4) dimethylation (H3K4me2), that contribute to the formation of a relaxed or “open” chromatin structure permissive for gene transcription (Surralles et al. 1998; Yan et al. 2003). Metal-related induction of acti- vating histone modifications have been sug- gested to contribute to metal carcinogenesis by causing the expression of tumor suppres- sor or other cancer-promoting genes (Wang et al. 2005; Zhou X et al. 2008). Inhaled PM pollutants have been previously shown to produce systemic changes in gene expres- sion, which can be detected in peripheral blood of exposed individuals (Salnikow and Zhitkovich 2008). However, whether metals in inhalable PM induce alterations of histone modifications in human subjects has never been evaluated. Although wide strata of the general popu- lation are exposed to low, background levels of toxic and carcinogenic inhalable compounds, industrial workers have been often exposed to significantly higher concentrations of poten- tially carcinogenic agents released from multi- ple sources (MacArthur et al. 2009). Foundry work is a specific condition of exposure to inhalable metal-rich PM that has been associ- ated with increased risk of lung cancer in sev- eral early investigations (International Agency for Research on Cancer 1987). Even in mod- ern foundry facilities, PM exhibits levels that are well above the concentrations found in ambient outdoor air and also have a larger pro- portion of toxic metal components (Alley et al. 2009; Fang et al. 2009; Roy et al. 2009; Vijay Bhaskar et al. 2009). In the present investiga- tion in a steel furnace plant, we investigated whether metals in inhalable PM determined changes in H3K4me2 and histone 3 lysine 9 acetylation (H3K9ac), two histone modifica- tions associated with open chromatin states, in peripheral blood leukocytes from workers with well-characterized exposure. Address correspondence to A. Baccarelli, Department of Environmental Health, Exposure, Epidemiology and Risk Program, Harvard School of Public Health, P.O. Box 15698, Boston, MA 02215 USA. Telephone: (617) 384-8742. Fax: (617) 384-8745. E-mail: abaccare@hsph.harvard.edu This work was supported by Cassa di Risparmio delle Provincie Lombarde (CARIPLO) Foundation grant 2007-5469 [Epigenetic Studies of Toxic Hazards in Environmental Research (ESTHER)]; Italian Ministry of Scientific Research, Research Programs of National Interest (grant PRIN 2007- 2S2HT8); and new investigator funding from the Harvard School of Public Health–National Institute of Environmental Health Sciences Center for Environmental Health (ES000002). The authors declare they have no actual or potential competing financial interests. Received 9 September 2010; accepted 8 March 2011. ovlume 119 | number 7 | July 2011 • Environmental Health Perspectives