Cantone et al. The changes observed in previous in vitro studies on lung carcinoma cells (Sun et al. 2009; Zhou X et al. 2009) and in our study in blood leukocyte samples suggest that induc- tion of gene-activating histone modifications such as H3K4me2 and H3K9ac might be a systemic process detectable across differ- ent tissues. However, whether H3K4me2 and H3K9ac are induced in human lung tis- sues exposed in vivo to carcinogenic metals needs to be confirmed in future investiga- tions. Metal components of inhaled PM have been shown to induce oxidative stress and inflammatory processes, which are known to affect histone modifications (Donaldson et al. 2003; Gilmour et al. 2003) and might specifi- cally affect blood leukocyte measures. A series of experimental studies using ambient PM10 collected in Utah Valley (USA) near a local steel plant demonstrated that anthropogenic PM containing bioavailable transition metals have heightened acute inflammatory effects, and that PM oxidant generation ability was enhanced in PM with higher metal content (Dye et al. 2001; Frampton et al. 1999; Ghio and Devlin 2001). Previous studies have shown that exposures to PM or related air- borne pollutants are associated with changes in proinflammatory and cancer-related gene expression in blood leukocytes (Feinberg and Tycko 2004; Franklin et al. 2008; MacNee and Donaldson 2003; Salnikow and Zhitkovich 2008) and with markers of gene expression control such as DNA methylation (Baccarelli et al. 2009; Bollati et al. 2007; Pavanello et al. 2009; Tarantini et al. 2009). We investigated a population with well- characterized exposure that allowed us to compare subjects over a wide range of dif- ferent exposure levels. We controlled for several potential confounders by fitting multi- variable models that included several indi- vidual characteristics as independent variables. However, we cannot exclude the possibility that other unmeasured exposures that are present in foundry facilities, such as heat, car- bon monoxide, and nonionizing radiation (Gomes et al. 2002; Lewis et al. 1992), might have influenced H3K4me2 and H3K9ac. In univariate analyses, we found that H3K9ac was positively associated with the education level of the study subjects. Although our study did not provide information to evaluate the biological basis for this association, our results showed that adjusted and unadjusted esti- mates for metal effects on histone modifica- tions were remarkably similar, so confounding from education and other variables included in our models is unlikely. Because of the limited number of study subjects, it is possible that the associations observed were attributable to chance. However, the occupational exposure and relatively con- trolled environment of a foundry provide a 968 good setting for evaluating these mechanistic questions and limit bias and chance findings. Our study was based on subjects working in several work areas of the same factory and did not include a different population of subjects without a specific condition of exposure to inhaled pollutants. Limiting our investigation to individuals who have all been working in the same work facility avoided potential con- cerns related to the selection of external refer- ents who might have differed from the exposed population in terms of socioeconomic factors and other characteristics determining hiring into the plant (Pearce et al. 2007). Nonetheless, the differences in the personal levels of expo- sure in our study group were large, providing sufficient contrast for identifying exposure- related changes in histone modifications. For example, the lowest level of PM10 observed in our study population (73.72 μg/m 3 ) was only marginally higher than ambient PM10 levels measured in the geographic area in which the plant is located (average annual ambient PM10 levels between 41 and 57 μg/m 3 were recorded in the year of the study by differ- ent ambient monitoring stations in Brescia area) (Anselmi and Patelli 2006), whereas the highest level was 1220.17 μg/m 3 . The sub- jects with the highest exposures to arsenic and nickel had exposure levels that were 36 and 31 times higher, respectively, than those for the least-exposed subjects. Although we based our study on a group of steel workers with aver- age exposures higher than that of the general population, the levels of exposure to metals in our study were all lower than the commonly accepted threshold limits for industrial set- tings (American Conference of Governmental Industrial Hygienists 2009). Because we determined differences in expo- sures within our study population by the dif- ferent tasks routinely performed by each of the study subjects, the personal exposure levels we measured in the week of the study reflected the usual exposure of the study subjects. We con- firmed this by the high correlation (r2 > 0.90) between exposure measures repeated over 3 years in a subset of the study population. Employment records showed that all the sub- jects included in the present study performed the same tasks for all the years they had been employed in the plant, suggesting that contrasts of exposure within the study population might have remained stable over time. Therefore, we estimated long-term cumulative exposure as the product of the levels of exposure by years of employment in the plant. However, because most of the study subjects had worked for more than a decade in the plant, it is possible that changes in production or exposure pro- tection regulations might have occurred over such an extended period of time. Therefore, we recognize that the estimated cumulative expo- sures we used in the present study are prone to exposure misclassification, and the results based on these metrics of exposure should be interpreted with caution. Conclusions Our results suggest that exposure to some metal components of PM, including nickel and arsenic, increased the two genes activating H3K4me2 and H3K9ac in blood leukocytes collected from healthy steel workers. Changes in the genomic levels of histone modifications may affect gene expression and contribute to the carcinogenic properties of inhalable nickel and arsenic. 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