Research Transcriptome Analysis in Peripheral Blood of Humans Exposed to Environmental Carcinogens: A Promising New Biomarker in Environmental Health Studies Danitsja M. van Leeuwen,1 Ralph W.H. Gottschalk,1 Greet Schoeters,2 Nicolas A. van Larebeke,3 Vera Nelen,4 Willy F. Baeyens,5 Jos C.S. Kleinjans,1 and Joost H.M. van Delft1 1Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, the Netherlands, 2Center of Expertise in Environmental Toxicology, Flemish Institute for Technological Research, Mol, Belgium; 3Study Centre for Carcinogenesis and Primary Prevention of Cancer, Ghent University Hospital, Ghent, Belgium, 4Provincial Institute of Hygiene, Antwerp, Belgium; 5Laboratory of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Brussels, Belgium BACKGROUND: Human carcinogenesis is known to be initiated and/or promoted by exposure to chemicals that occur in the environment. Molecular cancer epidemiology is used to identify human environmental cancer risks by applying a range of effect biomarkers, which tend to be nonspeciﬁc and do not generate insights into underlying modes of action. Toxicogenomic technologies may improve on this by providing the opportunity to identify molecular biomarkers consisting of altered gene expression proﬁles. OBJECTIVES: The aim of the present study was to monitor the expression of selected genes in a random sample of adults in Flanders selected from speciﬁc regions with (presumably) different envi- ronmental burdens. Furthermore, associations of gene expression with blood and urinary measures of biomarkers of exposure, early phenotypic effects, and tumor markers were investigated. RESULTS: Individual gene expression of cytochrome p450 1B1, activating transcription factor 4, mitogen-activated protein kinase 14, superoxide dismutase 2 (Mn), chemokine (C-X-C motif) lig- and 1 (melanoma growth stimulating activity, alpha), diacylglycerol O-acyltransferase homolog 2 (mouse), tigger transposable element derived 3, and PTEN-induced putative kinase1 were meas- ured by means of quantitative polymerase chain reaction in peripheral blood cells of 398 individu- als. After correction for the confounding effect of tobacco smoking, inhabitants of the Olen region showed the highest differences in gene expression levels compared with inhabitants from the Gent and fruit cultivation regions. Importantly, we observed multiple signiﬁcant correlations of particu- lar gene expressions with blood and urinary measures of various environmental carcinogens. CONCLUSIONS: Considering the observed signiﬁcant differences between gene expression levels in inhabitants of various regions in Flanders and the associations of gene expression with blood or uri- nary measures of environmental carcinogens, we conclude that gene expression proﬁling appears promising as a tool for biological monitoring in relation to environmental exposures in humans. KEY WORDS: biomarker, biomonitoring, environmental carcinogens, human blood, transcriptomics. Environ Health Perspect 116:1519–1525 (2008). doi:10.1289/ehp.11401 available via http://dx.doi.org/ [Online 23 June 2008] Cancer is among the major causes of death in modern society (Boyle and Ferlay 2005). Carcinogenesis is initiated and/or promoted by exposure to chemicals with carcinogenic properties that occur in the human environ- ment, for example, from ambient air, food, or lifestyle-related factors such as cigarette smoke (Czene et al. 2002; Higginson 1993; Le Marchand 2005; Luch 2005). Thus, in the- ory, a portion of cancer cases is preventable. Molecular epidemiology tools are used to iden- tify human cancer risks posed by exposure to environmental carcinogens, thereby contribut- ing to the scientific basis for environmental health policy measures. To this end, biomark- ers are investigated that enable human risk assessment long before traceable, diagnostic health effects appear. In monitoring environmental cancer risk among human populations, several markers of early biological effects [e.g., DNA damage markers such as micronuclei (MN) and DNA strand breaks] have been developed and applied over the last decades (Bonassi et al. 2005). However, these biomarkers tend to be nonspeciﬁc, raise questions about their sensitiv- ity, and do not generate insights in underlying modes of action. Genomic technologies such as microarrays and quantitative polymerase chain reaction (PCR) provide the opportunity to explore altered expression of large numbers of genes simultaneously (Afshari et al. 1999; Gerhold et al. 2001; Lobenhofer et al. 2001; Lockhart and Winzeler 2000) and therefore may provide the opportunity to identify molecular biomarkers consisting of altered gene expression proﬁles representing environmental health risks. Studies on carcinogen-induced dif- ferential gene expression have been conducted in our laboratory previously. Besides experi- mental in vitro studies that were directed toward obtaining gene expression proﬁles from model carcinogens in human blood mono- nuclear cells (van Leeuwen et al. 2005), a con- cise human population of smoking-discordant monozygotic twin pairs has been investigated; genes were identiﬁed of which the expression signiﬁcantly differed in smokers compared with their nonsmoking, genetically identical siblings (van Leeuwen et al. 2007). Furthermore, in a Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008 study of children from the Czech Republic, numerous gene expressions appeared relatively increased among children inhabiting a severely polluted area (van Leeuwen et al. 2006). From these studies, eight genes have been identified as promising biomarkers for envi- ronmental carcinogenesis. They encompass genes of which the expression differed signiﬁ- cantly between carcinogen-exposed and non- exposed individuals, in addition to genes that correlated significantly with an established biomarker of early biological effect (i.e., MN frequencies) (van Leeuwen et al. 2006, 2007). The aim of the present study was to monitor the expression of this set of genes in humans inhabiting speciﬁc regions in Flanders and to associate these with blood and urinary meas- ures of established biomarkers of exposure and early biological effect. We measured the expression levels of these eight key genes— cytochrome P450 1B1 (CYP1B1), activating transcription factor 4 (ATF4), mitogen- activated protein kinase 14 (MAPK14), super- oxide dismutase 2 (Mn) (SOD2), chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1), diacyl- glycerol O-acyltransferase homolog 2 (mouse) (DGAT2), tigger transposable element derived 3 (TIGD3), and PTEN-induced putative kinase-1 (PINK1) (van Leeuwen et al. 2006, 2007) (Table 1)—in peripheral blood cells by means of quantitative PCR. Furthermore, we explored associations with blood and urinary measures of biomarkers of Address correspondence to D.M. van Leeuwen, Department of Health Risk Analysis and Toxicology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, the Netherlands. Telephone: 31 (0)433882127. Fax: 31 (0)433884146. E-mail: d.vanleeuwen@grat.unimaas.nl We thank the volunteers for participating and the nurses for the sample collections. This study was conducted as part of the Flemish Centre of Expertise of Environment and Health and was commissioned, financed, and steered by the Ministry of the Flemish Community (Department of Economics, Science and Innovation; Flemish Agency for Care and Health; and Department of Environment, Nature and Energy). The sponsor was not involved in analysis or interpretation of the data. The authors declare they have no competing ﬁnancial interests. Received 21 February 2008; accepted 23 June 2008. 1519