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i I I I I I I 1 I I I I I I I I I I BIOASSAYS OF BENZO(A)PYRENE AND LUNG CANCER Wu Zhon -g liang*, Chen Jia-kun*, Zhan De jin*, Jin Bo*, He Ling*, Du Ying-xiu* and Joseph M. Wu** * Guangzhou Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou,China ** Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, USA Introduction Benzo(a)pyrene (B(a)P) is a ubiquitous environmental contaminant generated by combustion of substances such as coal, tobacco and other organic chemicals. 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 to B(a)P(l). B(a)P is a procarcinogen that requires metabolic activation to exert its mutagenic and carcinogenic effects(2,3). The metabolism of B(a)P has been studied in detail and the mutagenicity or carcinogenicity of B(a)P metabolites have been examined in a variety of prokaryotes, eukaryotes and experimental animals. These studies have shown that the amount and type of metabolic activity for biotransformation of B(a)P differs markedly 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 humans because of inter- and intra- species variability. In this study, human fetal broncho-epithelial cells (HFBE) cultured in vitro were used as an assay system to investigate the genotoxicity of the metaboltes of B(a)P for a better understanding of the role of B(a)P in human lung cancer initiation. Materials and Methods Materials Anti-7,8-dihydrodiol-9,10-epoxybenzo(a)pyrene (anti-BPDE), syn-7,8-dihydrodiol-9,10- epoxybenzo(a)pyrene(syn-BPDE), 9-hydroxybenzo(a)pyrene(9-OH-B(a)P), 3-hydroxybenzo(a)pyrene (3-OH-B(a)P) and 7,8-dihydrodiolbenzo(a)pyrene (7,8-diol-B(a)P) were purchased from commercial sources. MCDB 153 medium, restriction enzymes, reagents used for culturing broncho-tracheal epithelial cells and oncogene analysis were obtained from Sigma Chemicals Co. All other reagents were purchased in China. Cell cultures and preparation of liver and lung microsomes 1. Bronchoepithelial cell cultures. Tracheobronchial tissues from an abortive fetus were cut into small pieces (2x2 mm) and seeded onto cover glasses coated with rat-tail collagen. The coverslips were placed in tissue culture plates. Cells were cultured in MCDB 163 medium supplemented with 0.1% fetal bovine serum, insulin (10 µg/m1), epinephrine (10 µg/m]), 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 I

I was replaced twice weekly. 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. Following their identification by immunohistochemical staining, epithelial cells were 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 °lo sodium chloride solution and 50 ml of 50 mM sodium pyrophosphate. After the tissues were homogenized, the homogenate was centrifuged at 10,000 xg for 20 min. The supernatant was recentrifuged at 100,000 xg for 60 min. The pellated microsomes were stored at -70° C until ready for use. Microsomal protein was determined by the Lowry method(4). 3. Metabolism of B(a)P by microsomes The metabolism of B(a)P was studied in a 100 ml reaction mixture containing 50 mM Tris-HCI (pH 7.4), 0.3 mM magnesium chloride, 0.1 mM NADP+, 0.2 mM glucose-6-phosphate, 10 units of glucose-6-phosphate dehydrogenase, 100 mg of microsomal protein and 4 µM B(a)P. After shaking at 37° C for 60 min, the reaction was stopped by adding an equal volume of acetone. Materials in the organic phase were extracted twice with 1.5 volumes of ethyl acetate. 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 stored at -20° C or dissolved in methanol for analysis by HPLC. 4. Unscheduled DNA synthesis (UDS) The coverslips on which epithelial cells were growing were placed into liquid scintillation vials, treated with'"C-TdR (0.01 µCi/ml) for 72 hr., and then with'H-TdR (I µCi/ml) and B(a)P metabolites for an additional 24 hr. 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 as described by Fenech and Morley(5). The epithelial cells cultured on the coverslip were exposed to the metabolites of B(a)P and cytochalasin B (3 µg/ml) for 24 hr. Micronuclei were scored in cytokinesis-blocked binucleus cells. The significance of the results was tested with the Poisson distribution method. Determination of point mutation of Ha-ras oncoeenes To determine point mutations on the Ha-ras oncogene, the polymerase chain reaction was used to amplify H-ras specific sequences present in DNA extracted from cells treated with the B(a)P metabolite, anti-BPDE. After outgrowths of the cells bordering the explants 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 hr later. Cells were treated with anti-BPDE once a week for four weeks. Some cultures were subsequently treated I I I I I I I I I I I I '

I ' I I I I I I I I I I I I I I I I with 12-o-tetra-decanoyl-phorbol-13-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(6,7). 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 550 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 Hpa 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 B(a)P was metabolized by microsomes isolated from human fetal liver and lung cells. The result indicated that three derivaties of dihydrodiolbenzo(a)pyrene [9, 10- diol-B(a)P, 7, 8-diol-B(a)P, 4 5-diol-B(a)P], two metabolites of hydroxybenzo (a) pyrene [9-OH-B(a)P, 8-OH-B(a)P] and one product of quinonebenzo (a) pyrene [quinone-B(a)P] were formed upon incubation with microsomes from either human fetal liver or lung cells. I

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I I I I I I I I I I I I I I I I I Treatment of HFBE cells with metabolites of B(a)P [anti-BPDE, syn-BPDE, 7, 8-diol-B(a)P, 9- OH-B(a)P, 8-OH-B(a)Pj resulted in induction of unscheduled DNA synthesis (UDS) in a concentration- dependent manner. (Table 1). Similar results were obtained with the broncho-epithelial cells isolated from different individuals indicating that no significant inter-individual variation existed. (Table 2). Each metabolite of B(a)P, 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 B(a)P metabolites studied, anti-BPDE had the most significant effect on either UDS or micronucleus formation in HFBE cells. These results demonstrated that metabolites of B(a)P can induce lesions in DNA which subsequently resulted in unscheduled DNA synthesis. Table 1. UDS in IIFBE cells induced by anti-BPDE (relative radioactivity, 'HP`C) Concentration Cell (µg/ml) HT-11-a HT-11-22 HT-11-12A 0.00 1.00 ± 0.20 1.00 ± 0.97 1.00 t 0.13 0.125 1.34 f0.60 1.18 0.86 6 5 1.25 0.25 0.250 1.43 t 0.23 1.81 t 0.18 1.65 t 0.46 0.500 2.36 0.88 8 2.55t0.17* 0 2.09 0.60 0.650 3.27 t 0.79* 3.20 t 0.19* 2.50 t 0.37 0.800 4.44 f 1.75* 6.18 f 0.23** 5.42 t 1.63* 1.000 1.88 0.21 1 2.07 f0.18 1.89 0.63 3 8 ± SD *P<0.05 ** P<0.01 Table 2. UDS in the same HFBE cells induced by B(a)P Metabolites (relative radioactivity, 'H/14C) Cnncentrauon (Izg/mt) an6-BPDE 0.000 1.00 ± 0.13 0.125 0.250 0.500 0.800 t SD 1.25 t 0.25 1.66 t 0.46 2.09 t 0.60** 5.42 t 1.83** P<0 05 syn-BPDE 7,8-dinl-B(a)P 1.00 0.22 2 1.00 0.09 9 0.98 0.44 4 1.04 0.09 8 1.33 t 0.33 1.29 t 0.05 1.46 t 0.29* 2.48 f 0.29"* 3.52 t0 57 P<0.01 1.33 ± 0.01 9-0H-B(a)P 3-011-11(a)P 1.00 0.02 2 1.00 0.04 4 1.66 t 0.40 - 1.70 027 7 1.08 0.18 8 1.20 1.16 6 1.37 0.25 5 N 3.57 t 1.17* 1.78 t 0.15• ~ 3 V 00 W -5- j rn f N I

I I Table 3. Micronucleus formation in cytokinesis-blocked HFBE cells induced by B(a)P metabolites (%) Concentration` (ug/ml) ; anti-BPDE " svri-BPDE 9-OH-B(a)P 3-OH-B(a)P 4 4 4 9 8 23** 4 15* 39** 5 20** 50** 6 10 8 *P<0.05 **P<0.01 ***P<0.001 After being treated intermittently with anti-BPDE, HFBE cells showed no significant morphological changes. There were no cellular morphology changes characteristic of transformed phenotypes. The PCR-amplified H-ras oncogene fragments had a length of 145 bp including codon 12 of the H-ras oncogene. Two HPaII sites are present in wild type ras gene (one at the 25 bp position, the other at the 81 bp position) (Fig. 2), which could be lost as a result of mutations. Figure 2 shows that anti-BPDE induced point mutation at codon 12 of H-ras oncogene (Fig. 3) I I I I I I I I PlIMES 1 ~'CJS]tGTCGCAAAA]CGTTCi• l4WE[ C!'pGGGACACCQGlAGCAC]' Hpa I[ pem.r 1 / \ TD. 1] cca.v H-m. evcaC.ac / I ~]!!p I ~Ehp I pnna 2 I M62 TSe ®yyme Hn II cteavege ~~ PC& ampli6ed Ciegmenu of cT-ras ®mgene © a S ~a ra 's:+~ ~~ ~ "'~ u5g.3 Daa.vrm aC n ess ccd® 12 muted= uy Em u HFLF enaly®s DNA fnffi =evL ned enC-BPDF`¢mtai HFBE teIIa wm ampliCud . faa 40 eyekn with ~ ecd digestai wiih Hls IL FaDawmg digmCm[, xwft~ hagmmte wae eepm.amd thmugh eW/e paiYeac®da geL Cad= 12 mtIIffi$m ie mdi®trd by tye hepde O==ae®a'mg m i46 imne pnrse. ~nde vi®ule as 81 and 64 ®ee parze m~.>gmt wild type H-tm aIIrls Laoe 1 in P=ti~ omtvt '.mt}+ Pln®id PUCP.J 6.6, lan® 2®d ]0 ue FCR ymiuEy nwa nE®ei tn'EE ceTie. Ievm 3 is sr~darie, 250 agyx174. Hp II xeenietiGa ' :ngmmq lavea 6 Eo 9.nd ime 1 wca F'C8H pmfltuv fz>ffi veriwa a"'FHE I7AiA Cvtci with aad-EPDEM a-re. aodaa :2 mucnti®s ue eem m ttess la>va. -6- I I I I I I I

I I I I I I I I I I I I i I I I I I Discussion B(a)P is a procarcinogen which requires metabolic activation to exert its carcinogenic effect. Activation occurs mainly in the liver. Fourteen kinds of metabolites may be formed by the metabolism of B(a)P. The majority of them are "not toxic," only a few metabolites have very significant biological activity. Metabolism of B(a)P by the lung has not been reported so far. Microsomal proteins from human fetal liver and lung are found to metabolize B(a)P into its ultimate carcinogenic fotms. These data are similar to those previously reported for human bronchoepithelial cells(8-10). In situ metabolism in lung tissues may be important in the initiation of cancer at these sites. The epithelial cells are of particular interest, since they are the first to be in contact with environmental contaminants. The ability of lung tissues to activate B(a)P may therefore be an important factor in the induction of lung cancer resulting from inhalation of air pollutants containing B(a)P, such as, tobacco smoke, cooking fuel, etc. In previous experimental studies, animals and their cells were used to detect whether B(a)P metabolites had potential harmful effects to lung tissue. The extrapolation to actual human situation of carcinogenesis based on studies in experimental animals and cells presents complex challenges because of inter-species differences. Human cells were used in the present study to 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 B(a)P. The results showed that anti-BPDE had the most significant effect in inducing UDS and enhancing the micronucleus formation. This finding was consistent with that published previously(I1). Kapituluik et al.(12) found that syn-BPDE did not induce tumor in mice. Thus, it is reasonable to conclude that anti-BPDE is the main carcinogenic metabolite of B(a)P, while 3-OH-B(a)P, 9-OH-B(a)P and 7, 8-diol-B(a)P are simply metabolic intermediates of B(a)P which must 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 NZ of deoxyguanosine(13). 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 B(a)P(14). Binding of anti-BPDE to DNA may damage DNA and induce occurrence of UDS and MN. Anti-BPDE and Syn- BPDE are two metabolites of B(a)P which have different stereoscopic structures and possibly have differently biological effects. Since anti-BPDE has the most significant mutagenic effect in human cells, among the five metabolites of B(a)P, and mutagenesis is generally correlated 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(15). Point mutations in ras oncogene have been observed in human tumors of diverse origin and in a wide variety of carcinogen-induced animal tumors(16,17). These results further support the hypothesis that the ras oncogene is directly activated -7- I

I by mutations induced in the DNA damage introduced by B(a)P 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 at codon 12 was found, despite the fact cells showed no significant morphological change. The initiation of point mutation of oncogenes was earlier than the transformation in cell morphology. The 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. -8- I I I I I I I I I I I I I I I

I I I I . References Brislow, L, et al.; Amer. J. Publ. Health, 1954; 44: 177 2. Huberma, E, et al.; Pro. Nat. Acad. Sci. (Wash), 1976; 73:607 I 3. Wood, AW, etal.; J. Biol. Chem., 1976; 251:4882 I 4. Lowry, OH, et al.; J. Biol. Chem., 1951;193:265 5. Fenech, M & Morley, A A; Muta. Res., 1985; 147:29 I 6. Capon, DJ, eta l.; Nature, 1983; 302:33 1 7. Minoru, Tada, et al.; Cancer Res., 1990; 50:1121 8. Autrup, H, et al.; Int. J. Cancer, 1980;25:293 I 9. Mivhsrl, H, et al.; Chem-biol. Iterations, 1988; 64:281 10 H i CC l I P h 1 . arr s, , eta .; n at ogenesis 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 I 12. Kapitulnik, J, et al.; Cancer Res., 1978; Sept 38(9);2661-5 I 13. Veffery, AM.; Pharmac. Ther., 1985;28:237 14. Verina, DM, et al.; In: H.H Hlatt et al. eds, Origins of Human Cancer New York: Gold Spring I 15. Harbor Laboratory Press, 1977;PP639-658 Santos, E, et al.; Proc. Nat. Acad. Sci. USA, 1983;80:4679 I 16. Barbacid, M.; Ann. Rev. Biochem., 1987; 56:779 I 17. Guerrero, I, Pellicer, A; Mutat. Res. 1987; 185:293 I O 00 i 'V co , -9- ~ 0) I ol