Cartinogenesis vol.12 no.5 pp.777-781, 1991
Sodiinm mtoite-sttmiiiiilsiitedl metabolic adtnvatnomi off toeiniz©[ifl]pyireinie 7,8-dlMnydhrocMoIl m Mimam polymorplhioiniiiiiclear
Despina Constantin, Bengt JernstroW, Ian A.Cotgreave and Peter Moldeus Department of Toxicology, Karolinska Institutet, Box 60400, S-10401 Stockholm, Sweden 'To whom correspondence should be addressed
Introduction Polycyclic aromatic hydrocarbons (PAHs*) such as benzofa]pyrene (B[a]P) are produced by combustion processes and thus widely distributed environmental contaminants (1). Numerous studies have clearly demonstrated that several PAHs are potent carcinogens in experimental animals following their metabolism to reactive intermediates and subsequent DNA binding (for reviews, see 2,3). Epidemiological studies have strongly indicated that the environment is a significant factor in the incidence of cancer in humans (4). Thus, airborne pollutants such as PAHs may be a major contributing factor in the development of primary cancer in the respiratory tract (5,6). A dominant source of such inhaled carcinogens is cigarette smoke and >90% of bronchial carcinoma in humans is most probably caused by exposure to •Abbreviations: PAH, poh/cyclk aromatic hydrocarbon; B[a]P, benzo[a]pyrene; BP-7,8-diol, »ranj-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; BPDE, mjrti-7,8Klihydit)xy^,lOstimulated polymorphonuclear leukocytes (PMNs).The production of these tetraols implicates the intermediate formation of the corresponding *ra/w-7,8-dihydroxy-9,10epoxy-7,8-9,10-tetrahydrobenzo[a]pyrene (and-VPDE). A 2- to Mold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitritestimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonlum sulphate, a NADPHoxidase inhibitor, lowered the production of tetraols in PMAstimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from [^(-J-BP^.S-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism.
smoke (5,6). Cigarette smoke also contains in addition to a number of carcinogenic PAHs, trace of SO2 and relatively high concentrations of NO and NO2 (7). These gaseous components may be co-carcinogenic (6) with PAHs by enhancing, for instance, their metabolism and ultimate activation to reactive intermediates. This is compatible with the observation that the incidence of primary lung cancer among cigarette smokers seems to be increased in air-contaminated urban areas compared to less contaminated areas (8). It is well established that most carcinogens require metabolic activation to electrophilic intermediates, and subsequent covalent binding to DNA in order to exert tumorigenic activity. Alternative activation pathways have been demonstrated such as those dependent on cytochrome P450, various peroxidases, lipooxygenases and peroxyl radicals (9,10) The ultimate carcinogenic forms of PAHs are so called bayregion diol-epoxides (2,11). For instance B[a]P is metabolized to rratts-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) by the sequential action of cytochrome P450 and epoxide hydrolase followed by epoxidation at the 9,10-position to yield rra/«-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (reviewed in 2). This last step has been demonstrated to be carried out by cytochrome P450 (2), lipooxygenase (12), peroxidase (11) and by pathways dependent on formation of peroxyl radicals (9,10). Inhalation of nitrous gases such as NO2 is known to cause direct damage to various lung cell types and to alter metabolic function of the lung (13,14). Nitrogen dioxide dissolves readily into mucous and into epithelial cells throughout the lung and is rapidly distributed through the body via the pulmonary circulation (15). In experimental animals exposed to NO2 the major products detectable in blood and urine are nitrite and nitrate (15,16). Exposure to NO2 is associated with many toxicological effects (17) and it is possible that many of these effects, including co-carcinogenicity with PAHs, are mediated via nitrate or nitrite. In fact, the latter has been shown to exert a number of detrimental effects on physiological functions including the ability to act as a single electron donor in oxidation reactions involving catalase, peroxidases and hemoglobin (18,19). This ability of nitrite to participate in peroxidase-catalysed reactions may be of importance in the co-carcinogenic effects of NO2 with PAHs. In animals, attempts have been made to estimate the blood levels of nitrite after NO2 exposure and concentrations in the /iM range have been observed (16). To our knowledge, nitrite blood levels in humans after exposure to NO2 or nitrate/nitritecontaining foodstuffs have not yet been estimated. The aim of the present study was to establish if nitrite is able to enhance the peroxidase-dependent bioactivation of BP-7,8-diol to those DNA-binding intermediates thought to be crucial for tumour initiation. A cellular system consisting of human polymorphonuclear leukocytes (PMNs) was employed. These cells are rich in myeloperoxidase and are present in large numbers throughout the circulation and accumulate at sites of injury or inflammation.
D.Constantin et al.
Materials and methods Chemicals Tritiated BP-7,8-diol (1.69 Ci/moi) was purchased through the NCI Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO, USA). Unlabelled (+)- and (-)-enantiomers of BP-7,8-diol (purity > % % assayed by HPLQ and 7,10/8,9- and 7/8,9,10-tetraols were purchased through NCI Chemical Carcinogen Repository (Chemsyn Science Laboratories, Lenexa, KA, USA). Prior to use, tritiated BP-7,8-diol was diluted with unlabelled substrate to 705 mCi/mmol. Calf thymus DNA (type I), l was assayed by reversed-phase HPLC (Waters 5 n NOVA-Pak Radial C-18 column, 8 x 100 mm) with fluorescence detection as previously described by Romert et al. (22), except that the flow rate was 4 ml/min. In general, 200 11I samples obtained from die experiments with PMNs were analysed. The peak areas of the tetrads obtained experimentally were compared to standard curves constructed with authentic tetraol standards. Covalent binding of [3H](-)-BP-diol metabolites to calf thymus DNA and proteins in incubations with PMNs Leukocytes (10 x lO'/ml) were incubated for 30 min at 37°C with 10 /iM tritiated (-)-BP-7,8-diol, calf thymus DNA (1 mg/ml) and 1 or 5 mM sodium nitrite in a total volume of 3 ml PBS, pH 7.4. The reaction was initiated by the addition of 100 nM PMA. Sodium azide (1 or 10 mM) was added to the incubations where indicated. The incubation was terminated by sedimenting the cells by centrifugation at 2000 r.p.m. for 10 min. DNA in the supernatant was isolated and assayed as described by NordenskjOld et al. (23), except that the supernatant obtained after centrifugation was not treated with protease. The isolated DNA was finally dissolved in 10 mM Tris-HCl buffer, pH 7.4 and the amount estimated by UV-absorption at 260 run (23). In order to determine the covalent binding of [3H](-)-BP-7,8-diol metabolites to leukocyte proteins, the pellet obtained above was washed 10 times with acetone and then solubilized in 1 % sodium sarcosylate overnight at 4°C. The solubilized proteins were extracted five times with ethyl acetate and the protein content determined according to Peterson (24). The amount of radioactivity bound to DNA or protein was estimated by liquid scintillation counting. Statistical analyses Experiments were performed on three separate occasions and the results are expressed as means ± standard errors (SE). Groups of data were compared for significant differences using the nonpaired Mest.
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Fig. 1. Stimulation of tetraol formation by 1 and 5 mM sodium nitrite during the incubation of PMNs with (+)-BP-7,8-diol. The control incubation consisted of: 5 x 10* cells, and 10 /iM (+)-BP-7,8-diol in a total volume of 3 ml PBS, pH 7.4. The results are expressed as formation of 7,10/8,9-tetraol (pmol/ml). The values have been corrected by subtracting background values (14-23 pmol Tl/ml incubation) from the different time points. These background values represent the amount of tetraols recovered at zero time incubation. O , Control; O, control + 100 nM PMA; • , control + 1 mM NaNOjj D , control + 1 mM NaNOj + 100 nM PMA; A, control + 5 mM NaNOj; A , control + 5 mM NaNO, + 100 nM PMA. Each point is shown as the mean of three experimental values. Error bars represent ± SEM. The significance between the control and the incubations with 1 or 5 mM NaNO^ in PMA-stimulated leukocytes, after 30 min, is shown as *P < 0.05.
in
in
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20
0
10
20
0
10
20
RETENTION TIME (min) Fig. 2. HPLC analysis of products formed from (+)-BP-7,8-diol during incubation with PMNs in the absence (A) or presence (B) of 100 nM PMA and with both PMA and 1 mM NaNO? (C). Peak I, 7,10/8,9-tetraol; peak H, 7/8,9,10-tetraol; peak m, (+)-BP-7,8-diol.
Results Metabolism of (+)-BP-7,8-diol in PMNs The results of the incubation of human PMNs with (+)BP-7,8-diol and various co-additives are shown in Figure 1 and typical results from HPLC analysis of the tetraols formed are shown in Figure 2. In the absence of PMA the formation of tetraols after 30 min of incubation was limited and addition of nitrite had no stimulating effect. Conversely, in PMA-stimulated leukocytes, (+)-BP-7,8-diol was metabolized 2-fold more efficiently to yield — 100 pmol Tl/ml incubation. The presence of 1 or 5 mM nitrite increased die production of tetraols up to ~ 4-fold. The results clearly show that nitrite stimulates the metabolism of (+)-BP-7,8-diol in PMA-activated PMNs. Figure
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Preparation of human polymorphonuclear leukocytes Human PMNs were isolated from buffy coat, essentially using the method described by Trush et al (20). In brief, the buffy coat obtained from centrifuged blood (300 ml) was mixed with dextran-saline buffer (100 ml) and incubated in inverted syringes (50 ml) for 45 min, at 37°C. The upper layer containing leukocytes was collected and centrifuged at 1250 r.p.m. for 10 min, at 4°C. The pellet was resuspended in buffer containing 0.155 M NH4C1, 0.01 M NaHCO3 and 0.1 mM EDTA, pH 7.4 and centrifuged again as described above. The PMNs were washed to remove contaminating erythrocytes and resuspended in PBS supplemented with glucose (0.1 %) and the cells were counted in a haemocytometer. This procedure gave an average yield of — 1 x 106 PMNs per ml Wood.
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Activation of benzo{a]pyrene 7,8-dihydrodiol
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TIME(mln) Fig. 3. Effect of sodium azide (A) and diphenylene iodonium sulphate (B) on the incubation of PMA-stimulated PMNs with (+>-BP-7,8-diol and 1 mM NaNO2 (control incubation). The results are expressed as formation of 7,10/8,9-tetraol (pmol/ml). Each point is shown as the mean of three experimental values. Error bars represent ± SEM. The values have been corrected by subtracting background values (28-56 pmol Tl/ml incubation for A and 3 5 - 8 6 pmolTl/ml for B) from the different time points. These background values represent the amount of tetraols recovered at zero time incubation. (A) O, Control; A, control + 1 mM NaN3; A, control + 10 mM NaN3. (B) O, Control; A, control + 1 mM DPI.
Covalent binding of [3H](-)-BP-7,8-diol metabolites to DNA co-incubated with leukocytes DNA-binding was estimated by incubating tritiated ( - ) BP-7,8-diol with PMNs under various conditions. The results are given in Table II and show that binding to exogenous DNA only occurred in incubations containing PMA-stimulated leukocytes. Incubation of cells in the presence of nitrite caused a dose-dependent increase in the binding whereas addition of sodium azide reduced the binding to DNA. No binding to added calf thymus DNA occurred when (-)-BP-7,8-diol and nitrite were incubated with non PMA-activated PMNs. Discussion Trush et al. (20) demonstrated previously that PMA-activated PMNs metabolize racemic BP-7,8-diol to reactive intermediates that covalently bind to exogeneous DNA and induce mutations in bacteria. In addition, these investigators presented results that indicated the involvement of myeloperoxidase (MPO) and reactive oxygen species in the reaction. It is known that in response to a stimulus such as PMA a series of events take place in leukocytes including increased production of superoxide (a result of NADPH-oxidase activation) and hydrogen peroxide (25,26) and release of MPO from the azurophilic granules (27). The results obtained in the present study with PMA-activated leukocytes are in agreement with the findings of Trush et al. (20) except that no inhibition of BP-7,8-diol metabolism with sodium azide was
Table I. Covalent binding of [3H](-)-7,8-diol metabolites ito leukocyte proteins pmol bound/mg protein
Incubation mixture 1. 2. 3. 4. 5. 6.
Control Control Control Control Control Control
(no 1PMA) + + + +
1 1 1 5
mM mM mM mM
NaNO? NaNOj + 1 mM NaN3 NaNOj + 10 mM NaN3 NaNO2
5.75 18.8 45.3 18.4 15.3 78.8
± 1.24 ± 4.5 ± 18.7* ± 4.1 ± 3.5 ± 28.2*
The control reaction consisted of 10 jtM tritiated (-)-BP-7,8-diol, 100 nM PMA and 10 x 106 PMNs. The results are means ± SEM for cells from eight individual donors and those marked with an asterisk are significantly (P < 0.05) different from incubation mixture no. 2.
Table n. Binding of [3H]BP-7,8-diol metabolites to DNA coincubated with PMA-stimulated leukocytes Incubation mixture 1. 2. 3. 4. 5. 6.
Control (no PMA) Control Control + 1 mM NaNO, Control + 1 mM NaNC>2 + 1 mM NaN3 Control + 1 mM NaNQi + 10 mM NaN3 Control + 5 mM NaNOj
pmol bound/mg DNA 0.53 1.4 2.1 1.2 1.3 3.0
± 0.1 ± 0.3 db 0.3* ± 0.2 ± 0.2 ± 0.4»
The control incubation contained: 10 pM tritiated (-)-BP-7,8-diol, 100 nM PMA, 10 x 10* PMNs and 1 mg/ml calf thymus DNA. The results are expressed as mean ± SEM for cells from eight individual donors and those marked with an asterisk are significantly (P < 0.05) different from incubation mixture no. 2.
observed. The discrepancy may in part be explained by the different methods used for studying the activation of BP-7,8-diol. Whereas Trush and co-workers measured chemiluminescence associated with the intermediate formation of a 9,10-dioxetane of BP-7,8-diol [i.e. the excited 9,10-dialdehyde-derivative formed by decomposition of the dioxetane (28,29)] the production of 7/8,9,10- and 7,10/8,9-tetraols was estimated in the present study. The structure of the major tetraol formed, the 7,10/8,9-tetraol, strongly indicates the intermediate and exclusive formation of the a/tfj-diastereomer of BPDE rather than a 9,10-dioxetane (30,31). Thus, taken together the observations suggest that different pathways are involved in the metabolism of BP-7,8-diol 779
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3 shows the effect of sodium azide and diphenylene iodonium sulphate (DPI) on the nitrite-stimulated ( + )-BP-7,8-diol metabolism in PMA-activated leukocytes. For instance, after 30 min of incubation, 10 mM sodium azide decreased the amount of tetraols formed by 60% whereas inhibition of NADPH oxidase by DPI reduced the tetraol formation by - 7 0 % . Covalent binding of [3H](-)-BP-7,8-diol metabolites to leukocyte proteins PMA-stimulated leukocytes catalysed the covalent binding of products derived from tritiated (-)-BP-7,8-diol to cellular proteins (Table I). Addition of nitrite increased the binding by up to 4-fold in a dose-dependent manner. A pronounced reduction in the extent of binding occurred when sodium azide was present in the incubations. No significant binding was observed in nonstimulated PMNs in the presence of sodium nitrite.
D.Constantly et al.
In the experiments devoted to DNA- and protein-binding the (—)-BP-7,8-diol was generally used. No attempt was made in this study to identify the products covalently bound, however we assume that (+)-anti-BPDE is involved—although a contribution of dioxetane-derived adducts and adducts derived from other diolderivatives cannot be excluded. Several possible pathways may participate in the metabolism of BP-7,8-diol in leukocytes. These 780
include cytochrome P450 (34), lipooxygenases (12,35), and peroxidase-dependent pathways (20). At present it is impossible to ascribe the metabolism of BP-7,8-diol to a single metabolic pathway or to establish to what extent they contribute to the overall metabolism. In conclusion, nitrite stimulates the metabolism of BP-7,8-diol to tetraols and products that bind covalently to cellular proteins and DNA present outside the cells. The results clearly indicate the intermediate formation of carcinogenic ( + )-anti-BPDE. Inflammation in the lung, and thus leukocyte mobilization and activation, is associated with carcinogenesis. As proposed previously (20) it is possible that these cells may promote the carcinogenic process by contributing to the metabolism of carcinogens and increasing the actual concentration of harmful products in the vicinity of target cells. Acknowledgements Drs Anwer Rahimtula and Albrecht Seidel are greatly acknowledged for helpful discussions. This study was supported by grants from the Swedish Environmental Protection Board, the Swedish Tobacco Company and the Swedish Cancer Society.
References l.Baum.E.J. (1978) Occurrence and surveillance of polycyclic aromatic hydrocarbons. In Gelboin.H.V. and T'so.P.O.P. (eds), Polycyclic Hydrocarbons and Cancer. Vol. 1, Academic Press, New York, pp. 45—62. 2.Thakker,D.R., Yagi.H., Levin.W., Wood.A.W., Conney.A.H. and Jerina.D.M (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. In Anders, M.W. (ed.), Bioactivation of Foreign Compounds. Academic Press, Orlando, pp. 177-242. 3. Graslund,A. and Jemstrdm.B. (1989) DNA-interactkm: covalent DNA-adducts of benzo{a]pyrene 7,8-dihydrodiol 9,10-epoxides studied by biochemical and biophysical techniques. Q. Rev. Biophys., 22, 1-37. 4. Doll.R. and Peto.R. (1981) The causes of cancer; quantitative estimates of avoidable risks of cancer in United States today. J. Natl. Cancer Inst., 66, 1191-1308,. 5. US Surgeon General (1982) The Health Consequences of Smoking: Cancer. US Dept of Health and Human Services, Washington. 6. IARC (1986) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemical to Humans. Vol. 38. Tobacco Smoking. IARC, Lyon. 7. Zeller.W.J. and Schmahl.D. (1986) Atiologie des Bronchialkarzinoms. In Matthys.H. (ed.), Luftverunreinigungen und Atemwegserkrankungen beim Menschen. PMI-Verlag, Frankfurt, pp. 84-89. 8. Cancer (1984) Statens Offentliga Utredningar, 1984 67, pp. 95 and 314. Liber tryck Stockholm. 9. Mamett,L.J. (1987) Peroxyl free radicals: potential mediators of tumor initiation and promotion. Carcinogenesis, 8, 1365-373. 10. Marnett.L.J. (1990) Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates. Environ. Health Perspect., 88, 5-12. 11. Sims,P., Grover.P.L., Swaisland.A., Pal,K. and Hewer,A. (1974) MetaboUr activation of benzo[a]pyrene proceeds by a diol-epoxide. Nature, 252, 326-328. 12. Hughes.M.F., Charnulhrat.W., Mason.R.P. and Eling.T.E. (1989) Epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo{a]pyrene via a hydroperoxide-dependent mechanism catalyzed by lipooxygenases. Carcinogenesis, 10, 2075-2080. 13. Mustafa.M.G. and Tierny.D.F. (1978) Biochemical and metabolic changes in the lung with oxygen, ozone, and nitrogen dioxide. Am. Rev. Resp. Dis., 118, 1061-1090. 14. Morrow.P.E. (1984) Toxicological data on NO,: an overview. In MiUer.FJ. and Menzel.D.B. (eds), Fundamentals of Extrapolation Modeling of Inhaled Toxicants: Ozone, and Nitrogen Diozide. Hemisphere, Washington, DC, pp. 205-227. 15. Goklstein.E., GoUstein.F., Peek,N.F. and Parks.NJ. (1980) Absorption and transport of nitrogen oxides. In Lee.S.D. (ed.), Nitrogen Oxides and their Effects on Health. Ann Arbor Science, Ann Arbor, MI, pp. 143-160. 16. Kunimoto.M., Tsubone.H., Tsujii.N., Mochhate,K., Kaya.K., Shimojo.N. and Miura.T. (1984) Effects of nitrate and nitrite, chemical intermediates of inhaled nitrogen dioxide, on membrane components of blood cells of rats. ToxicxA. AppL Pharmacol., 74, 10-16. 17 Pool.B.L. Brendler.S., Klein,R.G., Monarca.S., Pasquini.R., Schmezer.P. and Zeller,W.J. (1988) Effects of SO2 or NOX on toxic and genotoxic
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in PMA-stimulated leukocytes; one that preferentially leads to anti-BPDE and one that results in a 9,10-dioxetane. The last pathway most probably involves participation of singlet oxygen (28,29,31). As evident from this study, sodium nitrite markedly stimulates the metabolism of (+)-BP-7,8-diol by PMA-activated leukocytes. Nitrite had no effect on the diol metabolism in non-activated leukocytes, suggesting an intimate association with the oxidative burst and/or related events such as the release of MPO from the azurophilic granules. The mechanism underlying the mode of action of nitrite upon the activation of BP-7,8-diol in PMAstimulated leukocytes is not understood. However, based on previous studies on the interaction of nitrite with haem-containing proteins (32) a possible mechanism can be proposed. By interacting with the ferric form of haem protein such as MPO, nitrite may act as an oxidizing agent in the reaction with BP-7,8-diol that results in intermediate formation of anti-BPDE. Sodium azide as well as DPI (inhibitors of MPO and NADPHoxidase respectively) inhibits the nitrite-stimulated metabolism of (+)-BP-7,8-diol in the leukocytes. At the concentration of DPI used here, the production of superoxide is known to be almost completely inhibited (33). However, as revealed by incubating the diol with a xanthine/xanthine oxidase system superoxide per se does not seem to be involved in the formation of tetraols (data not shown). The inhibiting effect of DPI on the metabolism of BP-7,8-diol seems to be indirect and possibly due to reduced formation of hydrogen peroxide and lower activity of MPO. The products formed from (+)- or (-)-BP-7,8-diol in PMAstimulated leukocytes in the presence or absence of nitrite included 7/8,9,10- and 7,10/8,9-tetraol as well as protein and DNAbinding intermediates. As mentioned previously, the results clearly indicate the intermediate and exclusive formation of antiBPDE [activation of ( + )- and (-)-BP-7,8-diol results in formation of ( - ) - and (+)-anti-BPDE respectively (30)] thus demonstrating a highly stereoselective reaction. In the experiments devoted to elucidating the metabolism of BP-7,8-diol the (+)-enantiomer was generally employed because this enantiomer has been shown to be a better substrate for the peroxidative metabolic pathway expected to contribute to or be fully responsible for the metabolism of (-)-BP-7,8-diol. On the other hand the more biologically important ( —)-enantiomer of BP-7,8-diol was used to study the covalent binding to DNA and proteins. Two independent pathways have been shown to participate in the metabolism of BP-7,8-diol to BPDE (9,10). The cytochrome P450 dependent metabolism of the (+)-enantiomer leads preferentially to (+)-syn-BPDE whereas the pathway involving haem-containing proteins in conjunction with a peroxide (e.g. lipid peroxide) preferentially results in (-)-anti-BPDE (9). The (-)-BP-7,8-diol on the other hand, may be metabolized by both pathways and results in the formation of ( + )-anti-BPDE, the ultimate carcinogenic form of B[a]P, and (-)-ryn-BPDE (2,30). The different pathways can be distinguished by HPLC analysis since the tetraols derived from syn- and anti-BPDE respectively are clearly separated under our conditions. In our experiments with (+)-BP-7,8-diol no tetraols compatible with the formation of (+)-.ryn-BPDE were observed.
Activation of benzo[a]pyrene 7,8-dihydrodiol
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properties of chemical carcinogens, n. Short term in vivo studies. Carcinogenesis, 9, 1247-1252. 18. Kosaka.H., Imaizumi.K. and Tyuma,I. (1981) Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite, an intermediate detected by electron spin resonance. Biochem. Bwphys. Acta, 702, 237-241. 19. Kosaka.H. and Tyuma.I. (1987) Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. Environ. Health Perspect., 73, 147-151. 20. Trush,M.A., SeedJ.L. and Kensler.T.W. (1985) Oxidant-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: Possible link between inflammation and cancer. Proc. Natl Acad. Sri. USA, 82, 5194-5198. 21. Green,M.J.,Hill,H.A. andTew.D.G. (1987) The rate of oxygen consumption and superoxide ankm formation by stimulated human neutrophils. FEBS Lett., 216, 31-34. 22. Romert,L. Dock,L., Jensscn,D. and Jernstr6m,B. (1989) Effects of glutathione transferase activity on benzo{a]pyrene-7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay. Carcinogenesis, 10, 1701-1707. 23. Nordenskjdld.M., Svensscm,S.-A., Jemstrom.B., Moldeus.P., Dock.L. and Soderhall.S. (1981) Studies on the in vitro transfer of DNA binding benzo(a]pyrene metabolites from rat hepatocytes to human fibroblasts. Carcinogenesis, 2, 1151 — 1169. 24. Peterson.G.L. (1977) A simplification of the protein assay method of Lowry which is more generally applicable. Anal. Biochem., 83, 346-356. 25. Babior.B.M. (1987) The oxidative burst oxidase. TIBS 12 June, 241 - 2 4 3 . 26. Bellavite.P. (1988) The superoxide-forming enzymatic system of phagocytes. Free Radical Biol. Med., 4, 225-261. 27. Wright,D.G. (1977) A functional differentiation of human neutrophil granules: generation of C5a by a specific (secondary) granule product and inactivation of C5a by azurophil (primary) granule products. J. Immunol, 119, 1068-1076. 28.Seligar,H.H., Thompson.A., Hamman.J.P. and Posner.G.H (1982) Chemihiminescence of benzo{a]pyrene-7,8-diol. Photochem. Photobioi., 36, 359-365. 29. Thompson.A., Biggley.W.H., Posner.G.H., LeverJ.R. and Seligar.H.H. (1986) Microsomal chemiluminescence of benzo(a)pyrene-7.8-dihydrodiol and its synthetic analogues arms- and cts-1-methoxyvinylpyrene. Biochem. Biophys. Acta, 882, 210-219. 30. Gelboin,H.V. (1980) Benzo[a]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes. Physiol. Rev., 60, 1107-1166. 31. Adam,W. and Cilento.G. (1983) Four-membered ring peroxides as excited equivalents: a new dimension in bioorganic chemistry. Angew. Chim. Int. Ed. Engi, 22, 529-542. 32. Young,L.J. and Siegel.L.M. (1988) On the reaction of ferric heme proteins with nitrite and sulfite. Biochemistry, 27, 2790-2800. 33. Cross,A R. (1987) The inhibitory effects of some iodonium compounds on the superoxide generating system on neutrophils and their failure to inhibit diaphorase activity. Biochem. Pharmacol, 36, 489-493. 34. Murray,G.I., Barnes.T.S., Scwell.H.F., Ewen.S.W.B., Melvin.W.T. and Burke.M.D. (1988) The immunocytochemical localization and distribution of cytochrome P-450 in normal human hepatic and extrahepatk tissues with a monoclonal antibody to human cytochrome P-450. Br. J. Clin. Pharmac., 25, 465-475. 35. Borgeat,P., Hamberg,M. and Samuelsson.B. (1976) Transformation of arachidonic acid and homo-6-linolenic acid by rabbit polymorphonuclear leukocytes. /. Biol. Chem,, 251, 7816-7820. Received on August 15, 1990; revised January 28, 1991; accepted on January 30, 1991
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Sodiinm mtoite-sttmiiiiilsiitedl metabolic adtnvatnomi off toeiniz©[ifl]pyireinie 7,8-dlMnydhrocMoIl m Mimam polymorplhioiniiiiiclear
Despina Constantin, Bengt JernstroW, Ian A.Cotgreave and Peter Moldeus Department of Toxicology, Karolinska Institutet, Box 60400, S-10401 Stockholm, Sweden 'To whom correspondence should be addressed
Introduction Polycyclic aromatic hydrocarbons (PAHs*) such as benzofa]pyrene (B[a]P) are produced by combustion processes and thus widely distributed environmental contaminants (1). Numerous studies have clearly demonstrated that several PAHs are potent carcinogens in experimental animals following their metabolism to reactive intermediates and subsequent DNA binding (for reviews, see 2,3). Epidemiological studies have strongly indicated that the environment is a significant factor in the incidence of cancer in humans (4). Thus, airborne pollutants such as PAHs may be a major contributing factor in the development of primary cancer in the respiratory tract (5,6). A dominant source of such inhaled carcinogens is cigarette smoke and >90% of bronchial carcinoma in humans is most probably caused by exposure to •Abbreviations: PAH, poh/cyclk aromatic hydrocarbon; B[a]P, benzo[a]pyrene; BP-7,8-diol, »ranj-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; BPDE, mjrti-7,8Klihydit)xy^,lOstimulated polymorphonuclear leukocytes (PMNs).The production of these tetraols implicates the intermediate formation of the corresponding *ra/w-7,8-dihydroxy-9,10epoxy-7,8-9,10-tetrahydrobenzo[a]pyrene (and-VPDE). A 2- to Mold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitritestimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonlum sulphate, a NADPHoxidase inhibitor, lowered the production of tetraols in PMAstimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from [^(-J-BP^.S-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism.
smoke (5,6). Cigarette smoke also contains in addition to a number of carcinogenic PAHs, trace of SO2 and relatively high concentrations of NO and NO2 (7). These gaseous components may be co-carcinogenic (6) with PAHs by enhancing, for instance, their metabolism and ultimate activation to reactive intermediates. This is compatible with the observation that the incidence of primary lung cancer among cigarette smokers seems to be increased in air-contaminated urban areas compared to less contaminated areas (8). It is well established that most carcinogens require metabolic activation to electrophilic intermediates, and subsequent covalent binding to DNA in order to exert tumorigenic activity. Alternative activation pathways have been demonstrated such as those dependent on cytochrome P450, various peroxidases, lipooxygenases and peroxyl radicals (9,10) The ultimate carcinogenic forms of PAHs are so called bayregion diol-epoxides (2,11). For instance B[a]P is metabolized to rratts-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) by the sequential action of cytochrome P450 and epoxide hydrolase followed by epoxidation at the 9,10-position to yield rra/«-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (reviewed in 2). This last step has been demonstrated to be carried out by cytochrome P450 (2), lipooxygenase (12), peroxidase (11) and by pathways dependent on formation of peroxyl radicals (9,10). Inhalation of nitrous gases such as NO2 is known to cause direct damage to various lung cell types and to alter metabolic function of the lung (13,14). Nitrogen dioxide dissolves readily into mucous and into epithelial cells throughout the lung and is rapidly distributed through the body via the pulmonary circulation (15). In experimental animals exposed to NO2 the major products detectable in blood and urine are nitrite and nitrate (15,16). Exposure to NO2 is associated with many toxicological effects (17) and it is possible that many of these effects, including co-carcinogenicity with PAHs, are mediated via nitrate or nitrite. In fact, the latter has been shown to exert a number of detrimental effects on physiological functions including the ability to act as a single electron donor in oxidation reactions involving catalase, peroxidases and hemoglobin (18,19). This ability of nitrite to participate in peroxidase-catalysed reactions may be of importance in the co-carcinogenic effects of NO2 with PAHs. In animals, attempts have been made to estimate the blood levels of nitrite after NO2 exposure and concentrations in the /iM range have been observed (16). To our knowledge, nitrite blood levels in humans after exposure to NO2 or nitrate/nitritecontaining foodstuffs have not yet been estimated. The aim of the present study was to establish if nitrite is able to enhance the peroxidase-dependent bioactivation of BP-7,8-diol to those DNA-binding intermediates thought to be crucial for tumour initiation. A cellular system consisting of human polymorphonuclear leukocytes (PMNs) was employed. These cells are rich in myeloperoxidase and are present in large numbers throughout the circulation and accumulate at sites of injury or inflammation.
D.Constantin et al.
Materials and methods Chemicals Tritiated BP-7,8-diol (1.69 Ci/moi) was purchased through the NCI Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO, USA). Unlabelled (+)- and (-)-enantiomers of BP-7,8-diol (purity > % % assayed by HPLQ and 7,10/8,9- and 7/8,9,10-tetraols were purchased through NCI Chemical Carcinogen Repository (Chemsyn Science Laboratories, Lenexa, KA, USA). Prior to use, tritiated BP-7,8-diol was diluted with unlabelled substrate to 705 mCi/mmol. Calf thymus DNA (type I), l was assayed by reversed-phase HPLC (Waters 5 n NOVA-Pak Radial C-18 column, 8 x 100 mm) with fluorescence detection as previously described by Romert et al. (22), except that the flow rate was 4 ml/min. In general, 200 11I samples obtained from die experiments with PMNs were analysed. The peak areas of the tetrads obtained experimentally were compared to standard curves constructed with authentic tetraol standards. Covalent binding of [3H](-)-BP-diol metabolites to calf thymus DNA and proteins in incubations with PMNs Leukocytes (10 x lO'/ml) were incubated for 30 min at 37°C with 10 /iM tritiated (-)-BP-7,8-diol, calf thymus DNA (1 mg/ml) and 1 or 5 mM sodium nitrite in a total volume of 3 ml PBS, pH 7.4. The reaction was initiated by the addition of 100 nM PMA. Sodium azide (1 or 10 mM) was added to the incubations where indicated. The incubation was terminated by sedimenting the cells by centrifugation at 2000 r.p.m. for 10 min. DNA in the supernatant was isolated and assayed as described by NordenskjOld et al. (23), except that the supernatant obtained after centrifugation was not treated with protease. The isolated DNA was finally dissolved in 10 mM Tris-HCl buffer, pH 7.4 and the amount estimated by UV-absorption at 260 run (23). In order to determine the covalent binding of [3H](-)-BP-7,8-diol metabolites to leukocyte proteins, the pellet obtained above was washed 10 times with acetone and then solubilized in 1 % sodium sarcosylate overnight at 4°C. The solubilized proteins were extracted five times with ethyl acetate and the protein content determined according to Peterson (24). The amount of radioactivity bound to DNA or protein was estimated by liquid scintillation counting. Statistical analyses Experiments were performed on three separate occasions and the results are expressed as means ± standard errors (SE). Groups of data were compared for significant differences using the nonpaired Mest.
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Fig. 1. Stimulation of tetraol formation by 1 and 5 mM sodium nitrite during the incubation of PMNs with (+)-BP-7,8-diol. The control incubation consisted of: 5 x 10* cells, and 10 /iM (+)-BP-7,8-diol in a total volume of 3 ml PBS, pH 7.4. The results are expressed as formation of 7,10/8,9-tetraol (pmol/ml). The values have been corrected by subtracting background values (14-23 pmol Tl/ml incubation) from the different time points. These background values represent the amount of tetraols recovered at zero time incubation. O , Control; O, control + 100 nM PMA; • , control + 1 mM NaNOjj D , control + 1 mM NaNOj + 100 nM PMA; A, control + 5 mM NaNOj; A , control + 5 mM NaNO, + 100 nM PMA. Each point is shown as the mean of three experimental values. Error bars represent ± SEM. The significance between the control and the incubations with 1 or 5 mM NaNO^ in PMA-stimulated leukocytes, after 30 min, is shown as *P < 0.05.
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20
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20
RETENTION TIME (min) Fig. 2. HPLC analysis of products formed from (+)-BP-7,8-diol during incubation with PMNs in the absence (A) or presence (B) of 100 nM PMA and with both PMA and 1 mM NaNO? (C). Peak I, 7,10/8,9-tetraol; peak H, 7/8,9,10-tetraol; peak m, (+)-BP-7,8-diol.
Results Metabolism of (+)-BP-7,8-diol in PMNs The results of the incubation of human PMNs with (+)BP-7,8-diol and various co-additives are shown in Figure 1 and typical results from HPLC analysis of the tetraols formed are shown in Figure 2. In the absence of PMA the formation of tetraols after 30 min of incubation was limited and addition of nitrite had no stimulating effect. Conversely, in PMA-stimulated leukocytes, (+)-BP-7,8-diol was metabolized 2-fold more efficiently to yield — 100 pmol Tl/ml incubation. The presence of 1 or 5 mM nitrite increased die production of tetraols up to ~ 4-fold. The results clearly show that nitrite stimulates the metabolism of (+)-BP-7,8-diol in PMA-activated PMNs. Figure
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Preparation of human polymorphonuclear leukocytes Human PMNs were isolated from buffy coat, essentially using the method described by Trush et al (20). In brief, the buffy coat obtained from centrifuged blood (300 ml) was mixed with dextran-saline buffer (100 ml) and incubated in inverted syringes (50 ml) for 45 min, at 37°C. The upper layer containing leukocytes was collected and centrifuged at 1250 r.p.m. for 10 min, at 4°C. The pellet was resuspended in buffer containing 0.155 M NH4C1, 0.01 M NaHCO3 and 0.1 mM EDTA, pH 7.4 and centrifuged again as described above. The PMNs were washed to remove contaminating erythrocytes and resuspended in PBS supplemented with glucose (0.1 %) and the cells were counted in a haemocytometer. This procedure gave an average yield of — 1 x 106 PMNs per ml Wood.
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Activation of benzo{a]pyrene 7,8-dihydrodiol
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TIME(mln) Fig. 3. Effect of sodium azide (A) and diphenylene iodonium sulphate (B) on the incubation of PMA-stimulated PMNs with (+>-BP-7,8-diol and 1 mM NaNO2 (control incubation). The results are expressed as formation of 7,10/8,9-tetraol (pmol/ml). Each point is shown as the mean of three experimental values. Error bars represent ± SEM. The values have been corrected by subtracting background values (28-56 pmol Tl/ml incubation for A and 3 5 - 8 6 pmolTl/ml for B) from the different time points. These background values represent the amount of tetraols recovered at zero time incubation. (A) O, Control; A, control + 1 mM NaN3; A, control + 10 mM NaN3. (B) O, Control; A, control + 1 mM DPI.
Covalent binding of [3H](-)-BP-7,8-diol metabolites to DNA co-incubated with leukocytes DNA-binding was estimated by incubating tritiated ( - ) BP-7,8-diol with PMNs under various conditions. The results are given in Table II and show that binding to exogenous DNA only occurred in incubations containing PMA-stimulated leukocytes. Incubation of cells in the presence of nitrite caused a dose-dependent increase in the binding whereas addition of sodium azide reduced the binding to DNA. No binding to added calf thymus DNA occurred when (-)-BP-7,8-diol and nitrite were incubated with non PMA-activated PMNs. Discussion Trush et al. (20) demonstrated previously that PMA-activated PMNs metabolize racemic BP-7,8-diol to reactive intermediates that covalently bind to exogeneous DNA and induce mutations in bacteria. In addition, these investigators presented results that indicated the involvement of myeloperoxidase (MPO) and reactive oxygen species in the reaction. It is known that in response to a stimulus such as PMA a series of events take place in leukocytes including increased production of superoxide (a result of NADPH-oxidase activation) and hydrogen peroxide (25,26) and release of MPO from the azurophilic granules (27). The results obtained in the present study with PMA-activated leukocytes are in agreement with the findings of Trush et al. (20) except that no inhibition of BP-7,8-diol metabolism with sodium azide was
Table I. Covalent binding of [3H](-)-7,8-diol metabolites ito leukocyte proteins pmol bound/mg protein
Incubation mixture 1. 2. 3. 4. 5. 6.
Control Control Control Control Control Control
(no 1PMA) + + + +
1 1 1 5
mM mM mM mM
NaNO? NaNOj + 1 mM NaN3 NaNOj + 10 mM NaN3 NaNO2
5.75 18.8 45.3 18.4 15.3 78.8
± 1.24 ± 4.5 ± 18.7* ± 4.1 ± 3.5 ± 28.2*
The control reaction consisted of 10 jtM tritiated (-)-BP-7,8-diol, 100 nM PMA and 10 x 106 PMNs. The results are means ± SEM for cells from eight individual donors and those marked with an asterisk are significantly (P < 0.05) different from incubation mixture no. 2.
Table n. Binding of [3H]BP-7,8-diol metabolites to DNA coincubated with PMA-stimulated leukocytes Incubation mixture 1. 2. 3. 4. 5. 6.
Control (no PMA) Control Control + 1 mM NaNO, Control + 1 mM NaNC>2 + 1 mM NaN3 Control + 1 mM NaNQi + 10 mM NaN3 Control + 5 mM NaNOj
pmol bound/mg DNA 0.53 1.4 2.1 1.2 1.3 3.0
± 0.1 ± 0.3 db 0.3* ± 0.2 ± 0.2 ± 0.4»
The control incubation contained: 10 pM tritiated (-)-BP-7,8-diol, 100 nM PMA, 10 x 10* PMNs and 1 mg/ml calf thymus DNA. The results are expressed as mean ± SEM for cells from eight individual donors and those marked with an asterisk are significantly (P < 0.05) different from incubation mixture no. 2.
observed. The discrepancy may in part be explained by the different methods used for studying the activation of BP-7,8-diol. Whereas Trush and co-workers measured chemiluminescence associated with the intermediate formation of a 9,10-dioxetane of BP-7,8-diol [i.e. the excited 9,10-dialdehyde-derivative formed by decomposition of the dioxetane (28,29)] the production of 7/8,9,10- and 7,10/8,9-tetraols was estimated in the present study. The structure of the major tetraol formed, the 7,10/8,9-tetraol, strongly indicates the intermediate and exclusive formation of the a/tfj-diastereomer of BPDE rather than a 9,10-dioxetane (30,31). Thus, taken together the observations suggest that different pathways are involved in the metabolism of BP-7,8-diol 779
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3 shows the effect of sodium azide and diphenylene iodonium sulphate (DPI) on the nitrite-stimulated ( + )-BP-7,8-diol metabolism in PMA-activated leukocytes. For instance, after 30 min of incubation, 10 mM sodium azide decreased the amount of tetraols formed by 60% whereas inhibition of NADPH oxidase by DPI reduced the tetraol formation by - 7 0 % . Covalent binding of [3H](-)-BP-7,8-diol metabolites to leukocyte proteins PMA-stimulated leukocytes catalysed the covalent binding of products derived from tritiated (-)-BP-7,8-diol to cellular proteins (Table I). Addition of nitrite increased the binding by up to 4-fold in a dose-dependent manner. A pronounced reduction in the extent of binding occurred when sodium azide was present in the incubations. No significant binding was observed in nonstimulated PMNs in the presence of sodium nitrite.
D.Constantly et al.
In the experiments devoted to DNA- and protein-binding the (—)-BP-7,8-diol was generally used. No attempt was made in this study to identify the products covalently bound, however we assume that (+)-anti-BPDE is involved—although a contribution of dioxetane-derived adducts and adducts derived from other diolderivatives cannot be excluded. Several possible pathways may participate in the metabolism of BP-7,8-diol in leukocytes. These 780
include cytochrome P450 (34), lipooxygenases (12,35), and peroxidase-dependent pathways (20). At present it is impossible to ascribe the metabolism of BP-7,8-diol to a single metabolic pathway or to establish to what extent they contribute to the overall metabolism. In conclusion, nitrite stimulates the metabolism of BP-7,8-diol to tetraols and products that bind covalently to cellular proteins and DNA present outside the cells. The results clearly indicate the intermediate formation of carcinogenic ( + )-anti-BPDE. Inflammation in the lung, and thus leukocyte mobilization and activation, is associated with carcinogenesis. As proposed previously (20) it is possible that these cells may promote the carcinogenic process by contributing to the metabolism of carcinogens and increasing the actual concentration of harmful products in the vicinity of target cells. Acknowledgements Drs Anwer Rahimtula and Albrecht Seidel are greatly acknowledged for helpful discussions. This study was supported by grants from the Swedish Environmental Protection Board, the Swedish Tobacco Company and the Swedish Cancer Society.
References l.Baum.E.J. (1978) Occurrence and surveillance of polycyclic aromatic hydrocarbons. In Gelboin.H.V. and T'so.P.O.P. (eds), Polycyclic Hydrocarbons and Cancer. Vol. 1, Academic Press, New York, pp. 45—62. 2.Thakker,D.R., Yagi.H., Levin.W., Wood.A.W., Conney.A.H. and Jerina.D.M (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. In Anders, M.W. (ed.), Bioactivation of Foreign Compounds. Academic Press, Orlando, pp. 177-242. 3. Graslund,A. and Jemstrdm.B. (1989) DNA-interactkm: covalent DNA-adducts of benzo{a]pyrene 7,8-dihydrodiol 9,10-epoxides studied by biochemical and biophysical techniques. Q. Rev. Biophys., 22, 1-37. 4. Doll.R. and Peto.R. (1981) The causes of cancer; quantitative estimates of avoidable risks of cancer in United States today. J. Natl. Cancer Inst., 66, 1191-1308,. 5. US Surgeon General (1982) The Health Consequences of Smoking: Cancer. US Dept of Health and Human Services, Washington. 6. IARC (1986) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemical to Humans. Vol. 38. Tobacco Smoking. IARC, Lyon. 7. Zeller.W.J. and Schmahl.D. (1986) Atiologie des Bronchialkarzinoms. In Matthys.H. (ed.), Luftverunreinigungen und Atemwegserkrankungen beim Menschen. PMI-Verlag, Frankfurt, pp. 84-89. 8. Cancer (1984) Statens Offentliga Utredningar, 1984 67, pp. 95 and 314. Liber tryck Stockholm. 9. Mamett,L.J. (1987) Peroxyl free radicals: potential mediators of tumor initiation and promotion. Carcinogenesis, 8, 1365-373. 10. Marnett.L.J. (1990) Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates. Environ. Health Perspect., 88, 5-12. 11. Sims,P., Grover.P.L., Swaisland.A., Pal,K. and Hewer,A. (1974) MetaboUr activation of benzo[a]pyrene proceeds by a diol-epoxide. Nature, 252, 326-328. 12. Hughes.M.F., Charnulhrat.W., Mason.R.P. and Eling.T.E. (1989) Epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo{a]pyrene via a hydroperoxide-dependent mechanism catalyzed by lipooxygenases. Carcinogenesis, 10, 2075-2080. 13. Mustafa.M.G. and Tierny.D.F. (1978) Biochemical and metabolic changes in the lung with oxygen, ozone, and nitrogen dioxide. Am. Rev. Resp. Dis., 118, 1061-1090. 14. Morrow.P.E. (1984) Toxicological data on NO,: an overview. In MiUer.FJ. and Menzel.D.B. (eds), Fundamentals of Extrapolation Modeling of Inhaled Toxicants: Ozone, and Nitrogen Diozide. Hemisphere, Washington, DC, pp. 205-227. 15. Goklstein.E., GoUstein.F., Peek,N.F. and Parks.NJ. (1980) Absorption and transport of nitrogen oxides. In Lee.S.D. (ed.), Nitrogen Oxides and their Effects on Health. Ann Arbor Science, Ann Arbor, MI, pp. 143-160. 16. Kunimoto.M., Tsubone.H., Tsujii.N., Mochhate,K., Kaya.K., Shimojo.N. and Miura.T. (1984) Effects of nitrate and nitrite, chemical intermediates of inhaled nitrogen dioxide, on membrane components of blood cells of rats. ToxicxA. AppL Pharmacol., 74, 10-16. 17 Pool.B.L. Brendler.S., Klein,R.G., Monarca.S., Pasquini.R., Schmezer.P. and Zeller,W.J. (1988) Effects of SO2 or NOX on toxic and genotoxic
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in PMA-stimulated leukocytes; one that preferentially leads to anti-BPDE and one that results in a 9,10-dioxetane. The last pathway most probably involves participation of singlet oxygen (28,29,31). As evident from this study, sodium nitrite markedly stimulates the metabolism of (+)-BP-7,8-diol by PMA-activated leukocytes. Nitrite had no effect on the diol metabolism in non-activated leukocytes, suggesting an intimate association with the oxidative burst and/or related events such as the release of MPO from the azurophilic granules. The mechanism underlying the mode of action of nitrite upon the activation of BP-7,8-diol in PMAstimulated leukocytes is not understood. However, based on previous studies on the interaction of nitrite with haem-containing proteins (32) a possible mechanism can be proposed. By interacting with the ferric form of haem protein such as MPO, nitrite may act as an oxidizing agent in the reaction with BP-7,8-diol that results in intermediate formation of anti-BPDE. Sodium azide as well as DPI (inhibitors of MPO and NADPHoxidase respectively) inhibits the nitrite-stimulated metabolism of (+)-BP-7,8-diol in the leukocytes. At the concentration of DPI used here, the production of superoxide is known to be almost completely inhibited (33). However, as revealed by incubating the diol with a xanthine/xanthine oxidase system superoxide per se does not seem to be involved in the formation of tetraols (data not shown). The inhibiting effect of DPI on the metabolism of BP-7,8-diol seems to be indirect and possibly due to reduced formation of hydrogen peroxide and lower activity of MPO. The products formed from (+)- or (-)-BP-7,8-diol in PMAstimulated leukocytes in the presence or absence of nitrite included 7/8,9,10- and 7,10/8,9-tetraol as well as protein and DNAbinding intermediates. As mentioned previously, the results clearly indicate the intermediate and exclusive formation of antiBPDE [activation of ( + )- and (-)-BP-7,8-diol results in formation of ( - ) - and (+)-anti-BPDE respectively (30)] thus demonstrating a highly stereoselective reaction. In the experiments devoted to elucidating the metabolism of BP-7,8-diol the (+)-enantiomer was generally employed because this enantiomer has been shown to be a better substrate for the peroxidative metabolic pathway expected to contribute to or be fully responsible for the metabolism of (-)-BP-7,8-diol. On the other hand the more biologically important ( —)-enantiomer of BP-7,8-diol was used to study the covalent binding to DNA and proteins. Two independent pathways have been shown to participate in the metabolism of BP-7,8-diol to BPDE (9,10). The cytochrome P450 dependent metabolism of the (+)-enantiomer leads preferentially to (+)-syn-BPDE whereas the pathway involving haem-containing proteins in conjunction with a peroxide (e.g. lipid peroxide) preferentially results in (-)-anti-BPDE (9). The (-)-BP-7,8-diol on the other hand, may be metabolized by both pathways and results in the formation of ( + )-anti-BPDE, the ultimate carcinogenic form of B[a]P, and (-)-ryn-BPDE (2,30). The different pathways can be distinguished by HPLC analysis since the tetraols derived from syn- and anti-BPDE respectively are clearly separated under our conditions. In our experiments with (+)-BP-7,8-diol no tetraols compatible with the formation of (+)-.ryn-BPDE were observed.
Activation of benzo[a]pyrene 7,8-dihydrodiol
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properties of chemical carcinogens, n. Short term in vivo studies. Carcinogenesis, 9, 1247-1252. 18. Kosaka.H., Imaizumi.K. and Tyuma,I. (1981) Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite, an intermediate detected by electron spin resonance. Biochem. Bwphys. Acta, 702, 237-241. 19. Kosaka.H. and Tyuma.I. (1987) Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. Environ. Health Perspect., 73, 147-151. 20. Trush,M.A., SeedJ.L. and Kensler.T.W. (1985) Oxidant-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: Possible link between inflammation and cancer. Proc. Natl Acad. Sri. USA, 82, 5194-5198. 21. Green,M.J.,Hill,H.A. andTew.D.G. (1987) The rate of oxygen consumption and superoxide ankm formation by stimulated human neutrophils. FEBS Lett., 216, 31-34. 22. Romert,L. Dock,L., Jensscn,D. and Jernstr6m,B. (1989) Effects of glutathione transferase activity on benzo{a]pyrene-7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay. Carcinogenesis, 10, 1701-1707. 23. Nordenskjdld.M., Svensscm,S.-A., Jemstrom.B., Moldeus.P., Dock.L. and Soderhall.S. (1981) Studies on the in vitro transfer of DNA binding benzo(a]pyrene metabolites from rat hepatocytes to human fibroblasts. Carcinogenesis, 2, 1151 — 1169. 24. Peterson.G.L. (1977) A simplification of the protein assay method of Lowry which is more generally applicable. Anal. Biochem., 83, 346-356. 25. Babior.B.M. (1987) The oxidative burst oxidase. TIBS 12 June, 241 - 2 4 3 . 26. Bellavite.P. (1988) The superoxide-forming enzymatic system of phagocytes. Free Radical Biol. Med., 4, 225-261. 27. Wright,D.G. (1977) A functional differentiation of human neutrophil granules: generation of C5a by a specific (secondary) granule product and inactivation of C5a by azurophil (primary) granule products. J. Immunol, 119, 1068-1076. 28.Seligar,H.H., Thompson.A., Hamman.J.P. and Posner.G.H (1982) Chemihiminescence of benzo{a]pyrene-7,8-diol. Photochem. Photobioi., 36, 359-365. 29. Thompson.A., Biggley.W.H., Posner.G.H., LeverJ.R. and Seligar.H.H. (1986) Microsomal chemiluminescence of benzo(a)pyrene-7.8-dihydrodiol and its synthetic analogues arms- and cts-1-methoxyvinylpyrene. Biochem. Biophys. Acta, 882, 210-219. 30. Gelboin,H.V. (1980) Benzo[a]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes. Physiol. Rev., 60, 1107-1166. 31. Adam,W. and Cilento.G. (1983) Four-membered ring peroxides as excited equivalents: a new dimension in bioorganic chemistry. Angew. Chim. Int. Ed. Engi, 22, 529-542. 32. Young,L.J. and Siegel.L.M. (1988) On the reaction of ferric heme proteins with nitrite and sulfite. Biochemistry, 27, 2790-2800. 33. Cross,A R. (1987) The inhibitory effects of some iodonium compounds on the superoxide generating system on neutrophils and their failure to inhibit diaphorase activity. Biochem. Pharmacol, 36, 489-493. 34. Murray,G.I., Barnes.T.S., Scwell.H.F., Ewen.S.W.B., Melvin.W.T. and Burke.M.D. (1988) The immunocytochemical localization and distribution of cytochrome P-450 in normal human hepatic and extrahepatk tissues with a monoclonal antibody to human cytochrome P-450. Br. J. Clin. Pharmac., 25, 465-475. 35. Borgeat,P., Hamberg,M. and Samuelsson.B. (1976) Transformation of arachidonic acid and homo-6-linolenic acid by rabbit polymorphonuclear leukocytes. /. Biol. Chem,, 251, 7816-7820. Received on August 15, 1990; revised January 28, 1991; accepted on January 30, 1991
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