Determination of Polycyclic Aromatic Hydrocarbons in the Urine, Benzo(A
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(CANCER RESEARCH 46, 4178-4183, August 1986] Determination of Polycyclic Aromatic Hydrocarbons in the Urine, Benzo(a)pyrene Diol Epoxide-DNA Adducts in Lymphocyte DNA, and Antibodies to the Adducts in Sera from Coke Oven Workers Exposed to Measured Amounts of Polycyclic Aromatic Hydrocarbons in the Work Atmosphere Aage Haugen,1 Georg Becher, Christel Benestad, Kirsi Vahakangas, Glennwood E. Trivers, Mark J. Newman, and Curtis C. Harris Department of Toxicology, National Institute of Public Health, 0462 OSLO 4, Norway [A. H., G. ft.]; Center for Industrial Research, P. O. Box 350, 0314 OSLO 3, Norway ¡C.B.J;Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 [K. V., G. E. T., C. C. H.]; and Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 ¡M.J. N.J ABSTRACT binding of reactive metabolites to DNA is considered to be an Workers in coke oven plants have a higher incidence of lung cancer important step in tumor initiation by these carcinogens (6). In than the general population. They are exposed to a variety of chemicals, fact, the carcinogenic potency of a number of PAH correlates in particular the polycyclic aromatic hydrocarbons (PAH), including with their ability to form specific covalent adducts with DNA benzo(a)pyrene. To evaluate the genotoxic effects of PAH exposure, air (6-8). However, the persistence of BPDE-DNA adducts per se samples and urine samples were analyzed for PAH by capillary gas is apparently not sufficient for tumor induction (9). BP, a chromatography and high-performance liquid chromatography, respec prototype ef the carcinogenic PAH and the most thoroughly tively. Since benzo(fl)pyrene is activated to 7/S,8a-dihydroxy-(9a,10a)- studied one, is metabolized to the chemically reactive isomers epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) and binds to DNA, we BPDE I and II, resulting in the formation of covalent BPDE- have used ultrasensitive enzymatic radioimmunoassay and synchronous adducts with DNA (5). fluorescence spectrophotometry to measure BPDE-DNA adducts in lym Sensitive methods are required to detect carcinogen-DNA phocyte DNA. The results show that workers were exposed to high concentrations of atmospheric PAH. However, the mean PAH exposure adducts in exposed individuals. Immunoassays, i.e., ELISA and USERIA (10-16), synchronous fluorescence spectrophotome levels are reduced 60% when the workers wore masks during work. When try (17-19), 32P-postlabeling assay (20), and HPLC 32P assay compared to exposure levels, the urinary excretion of PAH was relatively low. Approximately one-third of the workers had detectable putative (21 ) are methods currently being developed to detect the pres BPDE-DNA adducts in lymphocytes by ultrasensitive enzymatic radioim ence of carcinogen adducts in DNA. Recent reports have indi munoassay, and 10% of the samples had emission peaks at 379 nm by cated the presence of BPDE-DNA adducts in human lung tissue synchronous fluorescence spectrophotometry. The four most positive of patients with cancer of the lung and in human WBC from samples were the same in both of the assays. Antibodies to an epitope(s) occupationally exposed groups (15, 18, 19, 22). The purpose of on BPDE-DNA were found in the sera of approximately one-third of the this study was to quantify the carcinogenic exposure and bioa- workers. Detection of DNA adducts and antibodies to these adducts are vailability of PAH by analysis of airborne PAH, urinary PAH internal indicators of exposure to benzo(a)pyrene. levels, and the amounts of BP derivatives bound to DNA in peripheral blood lymphocytes of coke oven workers exposed to INTRODUCTION high levels of PAH in the work environment. In addition, we Emissions from coke ovens pose a significant risk of cancer examined the sera from these workers for antibodies to an epitope(s) on BPDE-DNA. to the exposed coke workers. This is based on epidemiological surveys (1, 2) as well as experimental animal studies (3) and MATERIALS AND METHODS chemical analyses of coke oven emissions (4). Epidemiological studies indicate an excess relative risk for lung cancer as high Chemicals. The solvents used were either "all glass distilled" in the as 16-fold for topside coke oven workers with 15 yr of exposure laboratory or HPLC grade from Rathburn, Ltd., Walkerbyrn, Scotland. or more (2). Chemical analyses of coke oven emissions have PAH standards were obtained from Fluka, Buchs, Switzerland revealed the presence of over 100 different PAH2 some of which (benz(a)anthracene, fluorene, and pyrene]; Koch-Light, Ltd., Suffolk, United Kingdom (benzo(a)fluorene, benzo(a)pyrene, benzo(e)pyrene, are known to be potent carcinogens in experimental animals and fluorene]; BDH, Ltd., Poole, United Kingdom (chrysene and fluor- (4). anthene); ICN Pharmaceuticals, Inc., Plainview, NY (2,2'-binaphthyl); PAH have long been of concern as a potential human health TCI, Tokyo, Japan (3,6-dimethylphenanthrene). hazard. There is considerable evidence that PAH are enzymat- Amberlite XAD-2 was obtained from Fluka, Buchs, Switzerland, and ically converted to reactive metabolites that bind covalently to cleaned by Soxhlet extraction with methanol, acetone, and cyclohexane cellular macromolecules (for review, see Ref. 5). The covalent prior to use. Proteinase (Pronase) was purchased from Calbiochem Corp. (La Jolla, CA) phenol (nucleic acid grade) from Bethesda Re Received 10/4/85; revised 4/11/86; accepted 4/30/86. search Laboratories, Inc. (Gaithesburg, MD); chloroform and isoamyl The costs of publication of this article were defrayed in part by the payment alcohol from J. T. Baker Chemical Co. (Phillisburg, NJ); alkaline of page charges. This article must therefore be hereby marked advertisement in phosphatase-conjugated goat anti-rabbit IgG (Fab2) from Cappel Lab accordance with 18 U.S.C. Section 1734 solely to indicate this fact. oratories, West Chester, PA; [3H]PNPP, Econofluor, from New Eng 1Present address: Department of Experimental Toxicology, Institute of Oc cupational Health. P. O. Box 8149 Dep, 0033 OSLO 1, Norway. To whom land Nuclear, Boston, MA; and PNPP from Sigma Chemical Co., St. requests for reprints should be addressed. Louis, MO. 2The abbreviations used are: PAH, polycyclic aromatic hydrocarbons; HP, Subjects. Topside coke oven workers (22 smokers, 8 exsmokers and benzo(a)pyrene; BPDE, 70,8a-dihydroxy-(9a, 1Oa)-epoxy-7,8,9,10-tetrahydro- benzo(u)pyrene; ELISA, enzyme-linked immunosorbent assay; PNPP, paranitro- 8 nonsmokers) were investigated. The age, sex, number of cigarettes phenyl phosphate; POM, polycyclic organic matter; SFS, synchronous fluores smoked per day, and medication were scored for all subjects. Urinary cence spectrophotometry; USERIA, ultrasensitive enzyme radioimmunoassay; PAH analyses were performed on 15 smokers and 9 nonsmokers and HPLC, high-performance liquid chromatography. DNA analyses on all 38 subjects. The urine and blood samples were 4178 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. BIOMONITORING OF COKE OVEN WORKERS collected at the end of the work shift. The urine samples were imme DNA as the solid phase competitor and to a well containing single- diately frozen, and blood samples were transported to the laboratory at stranded cold thymus DNA for specificity control. After 90-min incu 4°C.The serum for antibody studies was frozen at —70°C.Eighty% of bation at 37°Cthe plates were washed, and alkaline phosphatase- these workers wore a full-face respiratory protection system with air- conjugated goat anti-rabbit IgG (Fab2) (1:500) was added and incubated filtering mask. Exposure levels of PAH were determined by personal 1 h at 37°C.The plate was washed, 100 n\ of 10 to 20 pmol of tritiated air sampling both inside and outside the respirator worn by topside PNPP and 80 to 90 pmol of PNPP per well in 10 mM MgCI were workers. Four samples from different persons were collected using a added, and the plate was incubated for 4 h at 37°C.Finally, 20 n\ from sampling system consisting of a membrane filter (Versapor 800; Gel- each well were then diluted in 2 ml of buffer and mixed with 4 ml of man, Inc.) and a back-up section of Amberlite XAD-2 resins. The Econofluor 2, and the hydrolyzed [3H]PNP was separated in the organic sample volumes outside and inside the respirator were 0.449-0.480 m3 phase to measure radioactivity. and 0.905-0.943 m3, respectively. ELISA for Detection of Antibody to BPDE-DNA Adducts. Human Analysis of Airborne PAH. The paniculate PAH collected on the sera were tested for the presence of antibodies as described before (18). filter and the gaseous PAH adsorbed on the XAD-2 resins were ex Briefly, 20 ng of BPDE-modified DNA (test antigen) or unmodified tracted in a Soxhlet apparatus for 24 h with méthylènechloridecon DNA (control antigen) were attached by drying to the wells of polyvi- taining 2,2'-binaphthyl and 3,6-dimethylphenanthrene as internal nylchloride microtiter plates (Costar, Cambridge, MA). Human serum standards. The extracts were carefully concentrated to a smaller volume samples were tested using triplicate tests and 4 log; serial dilutions. under a gentle stream of highly purified N2. The PAH were transferred The binding of immunoglobin was detected using goat anti-human to freshly distilled cyclohexane and cleaned by liquid/liquid extraction immunoglobin reagents and the avidin-biotin horseradish-peroxidase with dimethyl formamide/water as described by Bj0rseth (23). The system (ABC Vectastain kit; Vector Laboratories, Burlingame, CA). PAH were analyzed by a Hewlett-Packard gas Chromatograph, Model The enzyme reaction was developed by the addition of 100 n\ of the 5730A, equipped with a SE-54 fused silica column and a flame ioniza- substrate solution [0.05 M citrate buffer (pH 4.0):o-phenylenediamine tion detector.