Short Communication Analysis of DNA and Hemoglobin Adducts and Sister Chromatid Exchanges in a Human Population Occupationally Exposed to Propylene Oxide: A Pilot Study

Propylene oxide (PO), a simple alkylating agent used in the chemical industry, is weakly genotoxic and induces nasal cavity tumors in rodents on inhalation at high air concentrations. DNA adducts, hemoglobin adducts, and sister chromatid exchanges (SCE) were analyzed as biomarkers of exposure in a group of eight PO-exposed workers and eight nonexposed subjects. 1-2Hydroxypropyladenine (1-HP-adenine) in DNA of WBCs was analyzed using a hypersensitive P-postlabeling assay. HP-valine in hemoglobin was measured using gas chromatography/tandem mass spectrometry. Air measurements indicated PO levels in the range of 1–7 ppm. All three biomarkers showed significantly increased levels in the exposed workers. 1-HP-adenine was recorded in seven of the exposed workers (mean 0.66 mol/10 mol nucleotides) but was not detected in any of the control subjects. HP-valine was found in all subjects (means of 2.7 and 0.006 pmol/mg globin in exposed workers and controls, respectively). The average frequencies of SCE were 3.7/cell in exposed workers and 2.0/cell in controls, respectively. DNA and hemoglobin adducts were correlated (r 0.887), as well as DNA adducts and SCE (r 0.792) and hemoglobin adducts and SCE (r 0.762). The present study is the first demonstrating PODNA adducts in human individuals. It is also the first study indicating cytogenetic effects in humans from PO exposure, although confounding effects from other sources cannot be excluded. Introduction PO is a common industrial chemical mainly used for production of propylene glycols, polyether polyols, and propylene glycol ethers. The Swedish exposure limit for PO is 2 ppm, and in the United States, the Occupational Safety and Health Administration has set a limit of 20 ppm, and the American Conference of Governmental Industrial Hygienists limit is set at 2 ppm. PO is weakly mutagenic and clastogenic and induces nasal cavity tumors in rodents on inhalation at high doses (1, 2). PO is a direct alkylating agent introducing HP groups on reactions with nucleophilic sites in DNA and proteins (2–4), and such adducts could therefore be used to monitor PO exposures (5). The major adduct formed in DNA after in vitro reaction of PO is 7-HP-guanine, followed by the adducts 3-HP-, 1-HP-adenine, and 3-HP-cytosine (3, 6). These adducts are chemically unstable: 7-HP-guanine and 3-HP-adenine will depurirate, forming apurinic sites, and 1-HP-adenine and 3-HPcytosine will spontaneously convert to N-HP-adenine and 3-HP-uracil, respectively (3, 6). PO adducts have been analyzed recently in rats exposed to PO by inhalation. 7-HP-guanine was analyzed by gas chromatography/mass spectrometry and by P-postlabeling (7, 8); the minor adducts have only been detected using the latter assay (6). Significant repair of either 7-HP-guanine or 1-HP-adenine was not seen in those animal studies. The level of 1-HP-adenine corresponded to 2% of 7-HP-guanine, but the recovery in the postlabeling assay exceeded that of 7-HP-guanine (50 versus 12%). In addition, 1-HP-adenine was chemically more stable (t1/2 9.2 days for rearrangement to an N adduct compared with t1/2 5 days for depurination of 7-HP-guanine; Ref. 6). Therefore, it was suggested that this minor adduct could be an alternative for monitoring PO exposures (6). Hemoglobin is the protein most commonly used for monitoring exposures to chemical carcinogens. PO adducts in hemoglobin are chemically stable, with the same life span as the erythrocytes themselves ( 4 months in humans). Hemoglobin adducts have been measured in humans after occupational exposure to PO (5, 9). DNA adducts from this compound have not been determined in human populations this far. Chromosomal aberrations, micronuclei, and unscheduled DNA synthesis have been analyzed in a human population exposed to PO (10–12). An effect on unscheduled DNA synthesis was observed, but the lack of a control population made it impossible to establish an effect of exposure for the cytogenetic data. The aim of the present study was, therefore, to measure both DNA and hemoglobin adducts and SCE in a small group of workers occupationally exposed to PO. Received 6/12/01; revised 11/28/01; accepted 12/14/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at the Department of Biosciences, Karolinska Institute, Novum, S-141 57 Huddinge, Sweden. Phone: 46-8-6089245; Fax: 46-8-6089501; E-mail: [email protected]. 2 The abbreviations used are: PO, propylene oxide; HP, 2-hydroxypropyl; SCE, sister chromatid exchange; HPLC, high-performance liquid chromatography. 315 Vol. 11, 315–318, March 2002 Cancer Epidemiology, Biomarkers & Prevention on June 22, 2017. © 2002 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Materials and Methods Sample Collection. Venous blood (5–10 ml) was collected from eight PO-exposed workers at a PO-producing plant and from an equal number of control subjects at an institute of occupational health in the Liaoning province, People’s Republic of China. The mean age was 29 (range 26–34) for the exposed group and 31 (range 26–33) for the control subjects. The individuals in the exposed group had worked 7–10 years in the factory, with 2 years with the present duties. There was an equal distribution of sexes, and there were two smokers in each group. PO air samples were collected on activated charcoal tubes for 20–60 min and analyzed by gas chromatography. Historical exposure measurements suggest that the levels were 10 ppm. Stationary air measurements recorded 1 day before blood sample collection revealed the highest levels in the packing area (3.7–6.9 ppm). In some common rooms and in the area of polymerization, the levels were 0.9–1.7 ppm. The workers spend 1–1.5 h/day in a packing area, transferring PO from larger reservoirs through pipes to containers of 100 liters and the rest of the 8-h working day in the common rooms not performing work duties related to PO or other exposures. Blood samples were collected in two heparinized glass tubes. One tube was frozen directly and used for later isolation of WBC DNA (7). The other tube was used for setting up the cell culture for cytogenetic analysis and isolation of globin. Adduct Analyses. 1-HP-adenine was analyzed by P-postlabeling as described (6). Postlabeled samples were mixed with the synthetically prepared standard 1-HP-5 -dAMP and separated by HPLC. The standard was detected by UV, and the peak was collected and pooled from four runs, each based on 10 g of DNA from the same person. The pooled sample was heated in 0.1 M NaOH (80°C, 30 min) and reanalyzed by HPLC with online UV and radioisotope detection. This treatment quantitatively converted 1-HP-5 -dAMP to N-HP-5 -dAMP. A DNA sample modified in vitro with C-labeled PO was used as an external standard to determine the recovery of 1-HP-adenine in the postlabeling assay (6). When analyzing this DNA, a recovery through the whole postlabeling procedure of 50% was obtained. The detection limit (after correcting for recovery) was 0.1 adduct/10 nucleotides. All human samples were coded before analysis, and each sample was analyzed at least twice. HP-valine was measured using the N-alkyl Edman method as described previously (13). All samples were coded before analysis. Hemoglobin was only obtained from six of the eight control individuals. Each sample was analyzed once. Previous studies have demonstrated a good reproducibility of the method with a SD of 5% of the mean. Cytogenetic Analysis. Each culture was set up by adding 0.5 ml of whole blood into 5 ml of culture medium containing F10 medium (Life Technologies, Inc., Breda, the Netherlands), 20% FCS (heat inactivated; Life Technologies, Inc.), heparin (32 units/ml; Sigma Chemical Co., St. Louis, MO), L-glutamine (1.65 mM; Life Technologies, Inc.), antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin), and phytohemagglutinin (0.3 mg/ml; Murex, Dartford, United Kingdom). 5-Bromo2 -deoxyuridine (BrdUrd, 10 M; Sigma Chemical Co.) was added, and the cultures were incubated for 72 h at 37°C in a 95% humidified incubator with 5% CO2. Air-dried metaphase preparations were made in a routine way. Sister chromatid differentiation using fluorescence plus Giemsa staining was not successful, indicating that the level of incorporated BrdUrd was low. Therefore, an anti-BrdUrd antibody was used for detecting SCEs. The slides were denatured for 1 min in 0.07 M NaOH and dehydrated in an ethanol series (70, 90, and 100%). Visualization of chromatids incorporated with BrdUrd was accomplished with anti-BrdUrd mouse antibody (Boehringer, Mannheim, Germany) and Alexa fluor 488 antimouse antibody (Molecular Probes, Eugene, OR) as described (14). Samples were coded before scoring.