Decreased levels of CXC-chemokines in serum of benzene-exposed workers identified by array-based proteomics

Abstract

Benzene is an important industrial chemical and environmental contaminant that causes leukemia. To obtain mechanistic insight into benzene's mechanism of action, we examined the impact of benzene on the human serum proteome in a study of exposed healthy shoe-factory workers and unexposed controls. Two sequential studies were performed, each using sera from 10 workers exposed to benzene (overall mean benzene air level >30 ppm) and 10 controls. Serum samples were subjected to anion-exchange fractionation and bound to three types of ProteinChip arrays (Ciphergen Biosystems, Fremont, CA) [hydrophobic (H50), metal affinity (IMAC3-Cu), and cation exchange (WCX2)]. Protein-expression patterns were detected by surface-enhanced laser desorption/ionization (SELDI)-TOF MS. Three proteins (4.1, 7.7, and 9.3 kDa) were consistently down-regulated in exposed compared with control subjects in both studies. All proteins were highly inversely correlated with individual estimates of benzene exposure (r > 0.75). The 7.7- and 9.3-kDa proteins were subsequently identified as platelet factor (PF)4 and connective tissue activating peptide (CTAP)-III. Initial proteomic results for PF4 and CTAP-III were subsequently confirmed in a single experiment using a ProteinChip-array-based immunoassay(Ciphergen Biosystems). The altered expression of the platelet-derived CXC-chemokines (40% and 63% for PF4 and CTAP-III, respectively) could not be explained by changes in absolute platelet counts. Thus, SELDI-TOF analysis of a limited number of exposed and unexposed subjects revealed that lowered expression of PF4 and CTAP-III proteins is a potential biomarker of benzene's early biologic effects and may play a role in the immunosuppressive effects of benzene.

Fig. 1.

RAI distributions for the three identified protein markers (m/z 4,057, protein A in Top; 7,763, protein B in Middle; and 9,287 protein C in Bottom) by test and exposure status (Boxplots, Left) and by benzene exposure (ppm) (Scatterplots, Right) for nonhemolyzed samples only (n = 34). (Left, Boxplots) The line within the box marks the median; the lower and upper boundary of the box indicates the 25th and 75th percentiles. Whiskers above and below the box indicate the 10th and 90th percentiles. (Right, Scatterplots) Scatter plots present RAI by benzene exposure (ppm) in the last month before blood collection. The solid line in the scatter plot represents the regression line between RAI and benzene exposure.

Fig. 2.

Spectra from ProteinChip array with immobilized antibodies against PF4 (B) and NAP-2 (C) for two exposed and two unexposed subjects and representative spectra of the negative control (nonspecific rabbit IgG; A). x axis depicts m/z. y axis depicts the average intensity of ion peaks.

Fig. 3.

Scatter plot and regression line between the RAI of the initial proteomic experiments and the PF4 and CTAP-III levels, as quantified by ProteinChip immunoassay (Ciphergen Biosystems). Open rounds are the results from the discovery set, and solid rounds are the results from the validation set of the initial experiment.