The study design, participants, environmental exposure assessment, and biological sample collection of this cross-sectional molecular epidemiological study have been previously described (Lan et al., 2010
). Briefly, a cross-sectional study of 80 workers currently exposed to TCE in six study factories with TCE cleaning operations and 96 unexposed controls were enrolled in June and July of 2006. Controls, frequency-matched by sex, and age (±5
years), were enrolled from two clothes manufacturing factories, one food production factory, and a hospital that did not use TCE, and were in the same geographic region as the factories that used TCE. For all potentially exposed workers and unexposed controls, subjects were excluded if they had a history of cancer, chemotherapy, and radiotherapy, as well as previous occupations with notable exposure to benzene, butadiene, styrene, and/or ionizing radiation. The study was approved by Institutional Review Boards at the U.S., National Cancer Institute and the Guangdong Poison Control Center in China. Participation was voluntary and all subjects gave written informed consent.
Personal air exposure measurements were conducted for all study subjects. Full-shift personal air exposure measurements were taken in the factories using 3
M organic vapor monitoring (OVM) badges before blood-collection. All samples were analyzed for TCE and a subset (48 from TCE-exposed workers) was analyzed for a panel of organic hydrocarbons including benzene, methylene chloride, perchloroethylene, and epichlorohydrin. OVM samples were also obtained on a subgroup of control workers. Subjects were categorized into three groups by mean TCE levels measured during the month before phlebotomy (unexposed workers, workers with <12
ppm TCE exposure, and workers with ≥12
ppm TCE exposure). A questionnaire-based interview, assessing demographics, lifestyle characteristics, and occupational history, was administered to all subjects.
After phlebotomy, lymphocyte subsets were analyzed on the same day that a peripheral blood sample was collected (Sallusto et al., 1999
). For the T lymphocyte subsets, peripheral white blood cells were preincubated with Fc block for 15
min at 4°C and immunostained with Abs recognizing the following: CD3 (UCHT1), CD4 (RPA-T4 or SK3), CD8 (SK1), CD45 (HI30), CD45RA (HI100), CCR7 (150503, R&D systems; all from BD Biosciences unless otherwise specified). Mouse IgG1 κ (MOPC-21) served as an isotype control. FCM data were acquired using a flow cytometer. For intracellular FoxP3 staining, peripheral white blood cells were first surface-stained with the following fluorescent Abs against CD4 (RPA-T4) and CD25 (M-A251). The cells were then fixed, permeabilized, and stained with anti-FoxP3 antibody using the Anti-Human Foxp3 Staining Set kit according to the manufacturer’s instructions (eBiosciences). Cells were then acquired and analyzed by FCM as described above. Measurements from blinded quality control replicates interspersed among the samples did not identify outlier batches. Assay CVs were <10% for each T cell lymphocyte subset analysis shown in Table , except for CD8+
central memory T cells which was 23%.
Unadjusted means and standard deviations were determined for each cell count subset. Linear regression using the natural logarithm (ln) of each subset was used to test for differences between unexposed and exposed workers, and to evaluate for a dose–response across exposure groups (unexposed workers, workers exposed to <12
ppm TCE, and workers exposed to ≥12
ppm TCE). All statistical models were adjusted for age (as a continuous variable) and sex. Potential confounders, including current cigarette smoking status (yes/no), current alcohol consumption (yes/no), recent infections (flu or respiratory infections in the previous month), and body mass index (BMI), were also included in a model for a specific subset if the regression coefficient was altered by ±15%. The total lymphocyte percent from the CBC was used to calculate lymphocyte subsets, and an additional calculation was carried out using the lymphocyte percent obtained by flow cytometry.