Toxicogenomics Article Discovery of Novel Biomarkers by Microarray Analysis of Peripheral Blood Mononuclear Cell Gene Expression in Benzene-Exposed Workers Matthew S. Forrest,1 Qing Lan,2 Alan E. Hubbard,1 Luoping Zhang,1 Roel Vermeulen,2 Xin Zhao,1 Guilan Li,3 Yen-Ying Wu,1 Min Shen,2 Songnian Yin,3 Stephen J. Chanock,2 Nathaniel Rothman,2 and Martyn T. Smith1 1School of Public Health, University of California, Berkeley, California, USA; 2Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA; 3National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China were then ranked and selected for further exam- Benzene is an industrial chemical and component of gasoline that is an established cause of ination using several forms of statistical analysis. leukemia. To better understand the risk benzene poses, we examined the effect of benzene expo- We also specifically examined the expression sure on peripheral blood mononuclear cell (PBMC) gene expression in a population of shoe- of all cytokine genes on the array under the factory workers with well-characterized occupational exposures using microarrays and real-time a priori hypothesis that these key genes polymerase chain reaction (PCR). PBMC RNA was stabilized in the field and analyzed using a involved in immune function are likely to be comprehensive human array, the U133A/B Affymetrix GeneChip set. A matched analysis of six altered by benzene exposure (Aoyama 1986). exposed–control pairs was performed. A combination of robust multiarray analysis and ordering We then attempted to confirm the array find- of genes using paired t-statistics, along with bootstrapping to control for a 5% familywise error ings for the leading differentially expressed rate, was used to identify differentially expressed genes in a global analysis. This resulted in a set genes using real-time PCR, which is thought to of 29 known genes being identified that were highly likely to be differentially expressed. We also be more accurate than microarray analysis but repeated these analyses on a smaller subset of 508 cytokine probe sets and found that the expres- can be used only to investigate a few genes at a sion of 19 known cytokine genes was significantly different between the exposed and the control time (Etienne et al. 2004). Once these genes subjects. Six genes were selected for confirmation by real-time PCR, and of these, CXCL16, were confirmed in the paired analysis, we ZNF331, JUN, and PF4 were the most significantly affected by benzene exposure, a finding that examined their expression in a larger number of was confirmed in a larger data set from 28 subjects. The altered expression was not caused by benzene-exposed subjects and controls. The changes in the makeup of the PBMC fraction. Thus, microarray analysis along with real-time overall goal is to provide potential gene markers PCR confirmation reveals that altered expressions of CXCL16, ZNF331, JUN, and PF4 are poten- of exposure and early effect for benzene and to tial biomarkers of benzene exposure. Key words: Affymetrix, benzene, biomarkers, blood, expres- produce mechanistic insight into how benzene sion profiling, leukemia, lymphocyte, microarray, molecular epidemiology, occupational exposure, affects the body, especially the immune system real-time PCR. Environ Health Perspect 113:801–807 (2005). doi:10.1289/ehp.7635 available via and lymphocyte function. http://dx.doi.org/ [Online 14 March 2005] Materials and Methods Study subjects and exposure assessment. We Benzene is an important industrial chemical gene expression in the peripheral blood leuko- studied workers exposed to benzene in two (> 2 billion gallons produced annually in the cytes of normal individuals (Whitney et al. shoe manufacturing factories and unexposed United States) and component of gasoline 2003). We hypothesized that microarrays could controls from three clothes manufacturing (Gist and Burg 1997). Its toxic effects on the identify changes in gene expression that could factories in the same region of Tianjin, blood and bone marrow include leukopenia, be used as new biomarkers of exposure and China. The study was approved by institu- pancytopenia, and aplastic anemia, and it is early effect for benzene and provide informa- tional review boards at all institutions. also an established cause of human leukemia tion on mechanisms of benzene toxicity. Participation was voluntary, written informed (Snyder 2002). However, the mechanisms of One potential problem with using consent was obtained, and the participation benzene-induced hematotoxicity and leukemo- microarrays in epidemiologic studies is that rate was approximately 95%. genesis remain unclear, as does the risk ben- mRNA is unstable (Thach et al. 2003). Most An initial group of six workers was selected zene poses at low levels of exposure (Krewski epidemiologic studies that have collected bio- from among the more highly exposed workers et al. 2000). To shed further light on these logic samples have not collected material that (mean benzene ± SD = 47.3 ± 24.3 ppm), and mechanisms and better understand the risk contains stabilized RNA for analysis. Here, we six controls were frequency-matched to these benzene poses, we examined the effects of have overcome this problem by performing the benzene exposure on peripheral blood first step of RNA isolation in the field and sta- Address correspondence to M.T. Smith, School of mononuclear cell (PBMC) gene expression in bilizing the RNA for later analysis. We have Public Health, 140 Warren Hall, University of a population of shoe-factory workers with analyzed this RNA from selected subjects using California, Berkeley, CA 94720-7360 USA. well-characterized occupational exposures to a comprehensive and standardized human Telephone: (510) 642-8770. Fax: (510) 642-0427. benzene using cDNA microarrays and real- array, the U133A/B Affymetrix GeneChip set E-mail: [email protected] time polymerase chain reaction (PCR). (Iacobuzio-Donahue et al. 2003). U133A and We thank H. Asahara for assistance with the Microarrays use immobilized cDNA or U133B chips together contain almost Affymetrix GeneChip hybridizations. This work was supported by National Institutes of oligonucleotide probes to simultaneously moni- 45,000 probe sets, representing > 39,000 Health grants RO1ES006721 and P30ES001896 to tor the expression of thousands of genes and unique transcripts derived from approximately M.T.S. obtain a view of global gene expression (i.e., a 33,000 well-substantiated human genes, allow- The authors declare competing financial interests. view of all mRNA transcripts expressed by a ing investigators to obtain a global view of M.T.S. has received consulting and expert testimony cell is known as the transcriptome) (Staudt gene expression. fees from law firms representing both plaintiffs and 2003; Staudt and Brown 2000) and are becom- We performed a proof-of-principle study in defendants in cases involving exposure to benzene. G.L. has received funds from the American Petroleum ing increasingly used in toxicology (Hamadeh which we examined global gene expression in a Institute for consulting on benzene-related health et al. 2002; Waters et al. 2003). They have also small number of well-matched exposed–control research. been used recently to investigate variation in subject pairs. Genes with differential expression Received 5 October 2004; accepted 14 March 2005. Environmental Health Perspectives • VOLUME 113 | NUMBER 6 | June 2005 801 Toxicogenomics | Forrest et al. subjects on the basis of age and sex. Mean age million PBMCs were added to 1 mL RLT 65°C for 5 min and then applied to U133B was 33.7 ± 7.1 years for the six exposed work- buffer (Qiagen, Valencia, CA) containing chips as described for U133A chips [of the ers and 31 ± 6.7 years for the controls. Four 1% β-mercaptoethanol to preserve RNA in the 45,000 probe sets, only 100 (which can be pairs were male and the other two female. cells. RNA that is frozen in this buffer at –80°C used for normalization) are found on both Before phlebotomy, individual benzene is highly stable. chips, so this “recycling” of hybridization cock- and toluene exposure was monitored by each RNA isolation, amplification, and tail should not affect the results]. Chips were wearing an organic vapor passive monitor hybridization. We isolated total RNA using then analyzed as described below. badge as previously described (Vermeulen RNeasy mini kits (Qiagen) according to manu- Chip normalization. To allow compari- et al. 2004). Personal full-shift air monitoring facturer instructions and quantified using a son, all chips were scaled to a target intensity was conducted about every month over a 3- to SmartSpec 3000 (Bio-Rad, Hercules, CA). of 500 based on all probe sets on each chip. 4-month period before biologic sample collec- Only samples with an A260/A280 between 1.7 Samples were run blind so that exposure sta- tion. Benzene and toluene were not detected and 2.2 were considered suitable for use. tus was unknown and designated as being in air samples from the control factories. Samples were prepared according to the either group A or B. Group A chips were used Each subject was given a physical exam GeneChip Eukaryotic Small Sample Target as the baselines when analyzing chips from by a study physician. A questionnaire was Labeling Assay Version II (Affymetrix 2003a), group B. administered that requested detailed informa- with the exception that the GeneChip Sample Statistical analysis to identify differen- tion on occupation, environmental exposures Cleanup Module (Affymetrix, Santa Clara, tially expressed genes. Robust multiarray analy- to solvents and pesticides, past and current CA) was used and not ethanol precipitation. sis (RMA) (Irizarry et al. 2003) was used to tobacco and alcohol use, past and current Total RNA (100 ng) was amplified for each analyze the data produced by the chips. Two medical history including recent infections, sample, with 400 ng of first-round cRNA RMA analyses of the GeneChip data were per- diagnostic and therapeutic ionizing radiation used for the second round of cDNA synthe- formed.