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BIOASSAYS OF BENZO(a)PYRENE AND LUNG CANCER Wu, Z.L.; Chen, J.K.; Zhan, D.J.; Jin, B.; He, L.; Du, Y.X., Wu,* J. :M . Guangzhou Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China. *Department of Biochemistry & Microbiology, New York Medical College, New York, 10595, USA. Introduction Benzo(a)pyrene [BaP] is a ubiquitous environmental contaminant associated with combustion of a number of substances such as coal, tobacco and other organic substances. Possible human exposure occurs through a number of routes including inhalation of polluted atmospheres and cigarette smoke. Epidemiological studies have shown a close relationship between human lung cancer and exposure I to BaPl". BaP is a procarcinogen that requires metabolic activation to exert its mutagenic and carcinogenic effects[2,31. Although the metabolism of BaP has been studied in detail and the mutagenicity or carcinogenicity of BaP metabolites have been examined in a variety of prokaryotes, eukaryotes and experimental animals, the amount and type of metabolic activity differs marked among species as well as among the tissues of a particular species. Thus, it is difficult to extrapolate the results from animals and cells to the human situation because of inter- and intra- species variability. In this i study, human fetal broncho-epithelial cells (HFBE) cultured in vitro were used as an assay system to investigate the genotoxicity of the metabolism of BaP for a better understanding of the role of BaP in human lung cancer initiation.

Materials and Methods Materials Anti 7,8-dihydrodiol-9, 10-epoxybenzo (a) pyrene (Anti-BPDE), syn 7, 8-dihydrodi6l-9, 10-epoxybenzo(a)pyrene (Syn-BPDE), 9-hydroxybenzo(a)pyrene[9-OH-BaP], 3-hydroxybenzo(a)pyrene [3-OH-BaP] and 7, 8-dihydrodiolbenzo(a)pyrene [7,8-diol-BaP] were graciously given to us by American friends. MCDB 153 medium, restriction enzymes and reagents used in broncho-tracheal epithelial cell cultures and oncogene analysis were obtained from Sigma Chemicals Co. All of the other reagents were purchased in China. Cell cultures and preparation of liver and lung microsome 1. Bronchoepithelial cell cultures. Tracheobronchial tissues from an abortive fetus were cut into small pieces (2x2 mm) and seeded onto cover glasses which were coated with collagen from rat tail. Then, the coverslips were placed in culture plates. MCDB 163 medium was used and supplemented with 0.1% fetal bovine serum, insulin (10 µg/ml), epinephrine (10 µg/ml), hydrocortisone (0.72 µg/ml), epidermal growth factor (2.0 µg/ml), transferrin (5 µg/ml) and antibiotics and incubated at 37° C in a humidified atmosphere of 5% CO2. The medium was replaced with fresh medium twice a week. When outgrowths of cells radiated from the tissues to a distance of 0.5 cm, repeated transfer of explants to new coverslips was done to reinitiated cell cultures. After being identified by - 2 -

immunohistochemical staining, epithelial cells on the original coverslip were ready to be used in this study. 2. Preparation of liver and lung microsomes. Liver and lung tissues from a fetus were cut into small pieces, rinsed with 0.9% sodium chloride solution and 50 ml 0.1m sodium pyrophosphate. After the tissues were homogenized, the homogenate was centrifuged (10,000 g, 20 min). The supernatant was centrifuged (10,000 g, 60 min) repeatedly. The precipitated microsomes were stored at -70° C until ready for use. Microsomal protein was determined by the Lowry method141. 3. Metabolism of BaP by microsomes. The evaluation of the aerobic metabolism of BaP was conducted in a 100 ml reaction mixture containing 10 mmol Tris-HC1 (pH 7.4), 0.3 mmol magnesium chloride, 0.1 mmol NADP+, 0.2 mmol glucose-6- phosphate, 10 units of glucose-6-phosphate dehydrogenase, 100mg of microsomal protein and 4 µmol BaP. After shaking, at 37° C for 60 min., the reaction was stopped by adding an equal volume of acetone. The organic soluble metabolites in the mixture were extracted with 1.5 volumes of ethyl acetate, twice. To stabilize the metabolites, 1% triethylamine was added to the ethyl acetate fraction. The organic phase was dried with anhydrous sodium sulphate and the solvent was evaporated under reduced pressure. The residue was dissolved in methanol for analysis by IIPLC or stored at -20° C. 4. Unscheduled DNA synthesis (UDS). - 3 -

Liquid-scintillation counting was conducted for UDS assay. The coverslips on which epithelial cells were growing were placed into liquid scintillation vials with MCDB 153 medium and treated with 14C-TdR (0.01 µCi/ml) for 72 hours. The medium was changed with a fresh one containing 2H-TdR (1 µCi/ml) and BaP metabolites for another 24 hours. The cells on the coverslip were washed with 0.9% saline solution and treated with trichloroacetic acid and absolute alcohol. After drying at 60° C, radioactivity was measured with a Beckman LS6000SC liquid scintillation system. 5. Micronucleus test. The method for the micronucleus test used in this study was described by Fenech and Morley~57. The epithelial cells cultured on the coverslip were exposed to the metabolites of BaP and cytochalasin B (3 ug/ml) for 24 hours. Micronuclei were scored in cytokinesis-blocking binucleus cells. The significance was tested with the Poisson distribution method. 6. Determination of point mutation of Ha-ras oncogenes. For determinating the point mutation of Ha-ras oncogenes, the polymerase chain reaction was used to amplify H-ras specific sequences of DNA extracted from cells treated with the BaP metabolite, anti-BPDE. After outgrowths of the cells bordering on the explants, which had been transferred to coverslips, radiated to a distance of 0.5 cm, anti-BPDE (1.5 µg/ml) was added to the medium. The medium was replaced by a fresh one 24 hours later. Cells were treated with anti-BPDE once a week for four weeks. Some - 4 -

cultures were subsequently treated with 12-o-tretra-decanoyl- phorbol-l3-acetate (TPA, 10 pg/ml) for two weeks. DNA was isolated from cells by standard techniques, and used as a template for PCR amplification of H-ras sequence. The PCR-primers used to amplify codon 12 of H-ras genes are shown in Fig 2~6,73 . PCR was performed at 97° C to denature the DNA for 5 min, at 72° C to anneal the primers for 1.5 min and at 93 and 55' C for 1 min at each temperature for primer extension. After amplification, H-ras point mutations were subsequently detected by the restriction fragment length polymorphism (RFLP) method with the use of the restricted enzyme Iipa II. The PCR product was digested with the restriction enzyme Hpa II. DNA fragments were electrophorezed on 6% polyacrylamide gel. Gels were stained with ethidium bromide and photographed on a UV transilluminator. Results HPLC analysis was performed after BaP was metabolized by microsome from human fetal liver and lung cells. The result indicated that three kinds of dihydrodiolbenzo(a)pyrene [9, 10- diol-BaP, 7, 8-diol-BaP, ,4 5-diol-BaP], two kinds of hydroxybenzo (a) pyrene [9-OH-BaP, 8-OH-BaP] and one type of quinonebenzo (a) pyrene [quinone-BaP] were formed after being metabolized by microsomes from either human fetal liver or lung cells. Figure 1 [To be added] When HFBE cells were treated with metabolites of BaP [anti- - 5 -

BPDE, syn-BPDE, 7, 8-diol-BaP, 9-OH-BaP, 8-OH-BaP], unscheduled DNA synthesis (UDS) could be induced. The magnitude of UDS was clearly dependent on the concentration of inetabolites. (Table 1). The same level of UDS existed after the broncho-epithelial cells from different individuals had been treated with these metabolites. This means that no significant inter-individual variation was present. (Table 2). Each metabolite of BaP, except for Syn-BPDE, could enhance the micronucleus rate of HFBE cells; it was evident that there was a dose-response relationship. (Table 3). The result described above show that among the BaP metabolites studied, anti-BPDE had the most significant effect on either UDS or micronucleus rate of HFBE cells. These results demonstrated that metabolites of BaP can induce lesions of DNA and subsequent unscheduled DNA synthesis. Table 1 Table 2 Table 3 After being treated intermittently with anti-BPDE, HFBE cells showed no significant morphological changes. There were no transformed characteristic changes in cellular morphology. The H-ras oncogene fragments which were amplified with PCR had the length of 145 bp including codon 12 of the H-ras oncogene. In wild type sequence of the ras gene there were two cleaving positions (CCGG) of restriction endonuclease Hpa II (one at the 25 bp position, the other at the 81 bp position) (Fig. 2). When - 6 -

mutations occur, this recognition site is lost. Figure 2 shows that anti-BPDE could induce point mutation at codon 12 of H-ras oncogene (Fig. 3) Figure 2 and 3 [To be added] Discussion BaP is a procarcinogen which requires metabolic activation to exert its carcinogenic effect. Activation occurs mainly in the liver. Fourteen kinds of inetabolites may be formed by metabolism of BaP. The majority of them are "not toxic," only a few metabolites have very significant biological activity. Metabolism of BaP by the lung has not been reported so far. Microsomal protein from human fetal liver and lung was used to metabolize BaP into its ultimate carcinogenic form, these data are similar to previously reported results on human bronchoepithelial cells[e,9,101. Tn situ metabolism in lung tissues may be important in initiation of cancer at these sites. The epithelial cells are of particular interest, since these cells are the first to be in contact with environmental contaminants. The ability of lung tissues to activate BaP may therefore be an important factor in the induction of lung cancer as a result of inhalation of air containing pollutants containing BaP, such as tobacco smoke, cooking fuel, etc. In previous experimental studies, animals and their cells were used to detect whether BaP metabolites had potential harmful - 7 -

effects to lung tissue. The extrapolation to the human situation of carcinogenesis studies in experimental animals and their cells presents complex problems because of inter-species differences. We used human cells in this study, which may avoid these shortcomings. Because the majority of human lung cancers originate from epithelial cells, it seems more reasonable to use human epithelial cells as target cells than animal cells or human fibroblasts. Using human epithelial cells may avoid inter-species differences and inter-tissue variability. Human fetal bronchoepithelial cells cultured in vitro were treated with each of the five metabolites of BaP. The results showed that anti-BPDE had the most significant effect in inducing UDS and enhancing the micronucleus rate. This finding was consistent with that of previous reportsC111. Kapituluik et al. found that syn-BPDE did not induce tumor in mice[121. Thus, it may be concluded that anti-BPDE is the main carcinogenic metabolite of BaP, while 3-OH-BaP, 9-OH-BaP and 7, 8-diol-BaP all are metabolic intermediates of BaP which may be metabolized further to form BPDE. Metabolic activation is the first step in the carcinogenesis process. Anti-BPDE can form a major DNA adduct by binding through its C10 position to the N2 of deoxyguanosine[133. It has been indicated that diol epoxide with the epoxyring located at the angular 'bay' region should be the most reactive, and therefore, likely to be the ultimate mutagenic and carcinogenic form of BaPI141. Binding of anti-BPDE to DNA may damage DNA and induce occurrence of UDS and MN. Anti-BPDE and Syn-BPDE are two - 8 -

metabolites of BaP which have different stereoscopic structures and which possibly have differently biological effects. Since Anti-BPDE has the most significantly mutagenic effect to human cells, among the five metabolites of BaP, and mutagenesis has some correlation with carcinogenesis, human fetal bronchoepithelial cells were treated continuously with anti-BPDE to further investigate its carcinogenesis by the determination of oncogene activation. The result indicated that cells grew normally and showed no morphological change. The point mutation at codon 12 of the H-ras oncogene in treated cellular DNA was detected by the polymerase chain reaction combined with RFLP analysis. It has been suggested that oncogenic activation occurs when any other aminoacid (except proline) is substitute in place of glycine as a result of a mutation in codon 12 of the ras gene[153. Point mutation in ras oncogene have been observed in human tumors of diverse origin and in a wide variety of carcinogen-induced animal tumors[16,173. These results further support the hypothesis that the ras oncogene can be directly activated by the mutations resulting from the DNA damage induced by BaP metabolites. Among human lung tumors, point mutations of ras oncogenes may exist in 50% of lung adenocarcinomas. Most of point mutations are also at codon 12. This indicates that point mutation of ras oncogenes at codon 12 has a close relationship with the initiation of lung cancer. In our transformation test of human bronchoepithelial cells, the point mutation of the H-ras oncogene - 9 -

at codon 12 was found, but cells showed no significant morphological change. The initiation of point mutation of oncogenes was earlier than the transformation in cell morphology which does not occur at an early stage of the test, so point mutation of oncogenes may be regarded as a sensitive indicator of cell transformation or an early stage of chemical carcinogenesis in human lung cancer. References 1. Brislow L, et al. amer J Publ Health, 1954; 44: 177 2. Huberma E, et al. Pro natt Acad Sci (wash), 1976; 73:607 3. Wood AW, et al. J. Biol Chem, 1976; 251:4882 4. Lowry OH, et al. J. Biol chem, 1951;193:265 5. Fenech M & Morley A A, Muta Res, 1985; 147:29 6. Capon DJ, et al. Nature, 1983; 302:33 7. Minoru Tada, et al. Cancer Res, 1990; 50:1121 8. Autrup H, et al. Int J Cancer, 1980;25:293 9. Mivhsrl H, et al. Chem-biol Iterations, 1988; 54:281 10. Harris CC, et al. In Pathogenesis an therapy of Lung Cancer, Harris, New York, Marcel Decker Inc, 1987;550 11. Heflich RN, et al. Biochem biophys REs Commum, 1977;77:634 12. Kapitulnik J, et al. Cancer Res, 1978; Sept 38(9);2661-5 13. Veffery AN. Pharmac Ther, 1985;28:237 14. Verina DM, et al. In: H.I-i Hlatt et al eds, Origins of Human Cancer New York: Gold Spring Harbor Laboratory Press, - 10 -